Amyloid Beta 1-6 Antigen Arrays

ABSTRACT

The present invention is related to the fields of molecular biology, virology, immunology and medicine. The invention provides a composition comprising an ordered and repetitive antigen or antigenic determinant array, and in particular an Aβ1-6 peptide-VLP-composition. More specifically, the invention provides a composition comprising a virus-like particle and at least one Aβ1-6 peptide bound thereto. The invention also provides a process for producing the conjugates and the ordered and repetitive arrays, respectively. The compositions of the invention are useful in the production of vaccines for the treatment of Alzheimer&#39;s disease and as a pharmaccine to prevent or cure Alzheimer&#39;s disease and to efficiently induce immune responses, in particular antibody responses. Furthermore, the compositions of the invention are particularly useful to efficiently induce self-specific immune responses within the indicated context.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a non provisional of U.S. ProvisionalApplication Nos. 60/396,639, filed Jul. 19, 2002; and 60/470,432, filedMay 15, 2003; both of which applications are entirely incorporated byreference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to the fields of molecular biology,virology, immunology and medicine. The invention provides a compositioncomprising an ordered and repetitive antigen or antigenic determinantarray, and in particular an Aβ1-6 peptide-VLP-composition. Morespecifically, the invention provides a composition comprising avirus-like particle and at least one Aβ1-6 peptide bound thereto. Theinvention also provides a process for producing the conjugates and theordered and repetitive arrays, respectively. The compositions of theinvention are useful in the production of vaccines for the treatment ofAlzheimer's disease and as a pharmaccine to prevent or cure Alzheimer'sdisease and to efficiently induce immune responses, in particularantibody responses. Furthermore, the compositions of the invention areparticularly useful to efficiently induce self-specific immune responseswithin the indicated context.

2. Related Art

Alzheimer's disease (AD) is the most common cause of dementia among theelderly (age 65 and older) and a serious burden for public health. Forexample, 4 million people are reported to suffer from the disease in theUnited Sates of America. The incidence of the disease is expected toincrease as the population ages.

The main pathological signs of Alzheimer's disease are age-relatedchanges in behaviour, deposition of β-amyloid into insoluble plaques,called the neuritic plaques or AD plaques, neurofibrillary tanglescomposed of tau protein within neurons, and loss of neurons throughoutthe forebrain. In addition to the late onset AD, which occurs in old age(65 years and more), there is an early onset AD, familial AD (FAD)occurring between age 35 and 60. The pathological abnormalities of ADare more widespread, severe and occur earlier in FAD than in late onsetor sporadic AD. Mutations in the APP gene, the presenilin 1 and thepresenilin 2 genes have been correlated with FAD.

As indicated, one of the key events in Alzheimer's Disease (AD) is thedeposition of amyloid as insoluble fibrous masses (amyloidogenesis)resulting in extracellular neuritic plaques and deposits around thewalls of cerebral blood vessels (for review see Selkoe, D. J. (1999)Nature, 399, A23-31). The major constituent of the neuritic plaques andcongophilic angiopathy is amyloid β (Aβ), although these deposits alsocontain other proteins such as glycosaminoglycans and apolipoproteins.Aβ is proteolytically cleaved from a much larger glycoprotein known asAmyloid Precursor Protein (APP), which comprises isoforms of 695-770amino acids with a single hydrophobic transmembrane region. Aβ forms agroup of peptides up to 43 amino acids in length showing considerableamino- and carboxy-terminal heterogeneity (truncation) as well asmodifications (Roher, A. E., Palmer, K. C., Chau. V., & Ball. M. J.(1988) J. Cell Biol. 107, 2703-2716. Roher, A. E., Palmer, K. C.,Yurewicz. E. C., Ball, M. J., & Greenberg, B. D. (1993) J. Neurochem.61, 1916-1926). Prominent isoforms are Aβ1-40 and 1-42. It has a highpropensity to form 8-sheets aggregating into fibrils, which ultimatelyleads to the amyloid.

Aβ peptide has a central role in the neuropathology of Alzheimersdisease. Region specific, extracellular accumulation of Aβ peptide isaccompanied by microgliosis, cytoskeletal changes, dystrophic neuritisand synaptic loss. These pathological alterations are thought to belinked to the cognitive decline that defines the disease.

Administration of amyloid beta protein or, in particular, Aβ 1-28 inamounts of up to 10⁻² mg/dose in the absence of any adjuvants andwithout any linkage of the amyloid beta protein or Aβ 1-28 to a carrier,for the treatment of Alzheimer's disease, is described in EP 526,511.

Others have used administration of Aβ peptides in combination withadjuvants, to induce an immune response, cellular or humoral, against Aβ1-42. In a transgenic mouse model of Alzheimer disease, animalsoverexpress human amyloid precursor protein containing the mutationAPP(717)V-F (PDAPP-mice; Johnson-Wood, K. et al., Proc. Natl. Acad. USA94: 1550-1555, Games, D. et al., Nature 373: 523-527 (1995a)), leadingto overproduction of Aβ₁₋₄₂, develop plaques, dystrophic neuritis, lossof presynaptic terminals, astrocytosis and microgliosis. In a recentstudy, Schenk, D. et al., (Nature 400:173-77 (1999) and WO 99/27944)report that administration of aggregated Aβ₁₋₄₂ mixed with a strongadjuvant (CFA/IFA), which cannot be used in humans, in the first 4immunizations, followed by administration of aggregated Aβ1-42 in PBS inthe subsequent immunizations, to PDAPP-mice at 6 weeks of age,essentially prevented plaque formation and associated dystrophicneuritis. The same authors reported that immunization of older mice (11months of age) using the same strategy markedly reduced the extent andprogression of Alzheimer's disease (AD)-like neuropathologies.Proliferation of splenocytes from mice immunized using theabove-mentioned strategy was reported in Example III (Screen fortherapeutic Efficacy against established AD) of WO 99/27944, showingthat Aβ1-42 specific T-cells were induced by the vaccination procedure.Coupling of Aβ fragments to sheep anti-mouse IgG, and immunization ofsaid coupled fragment in the presence of the adjuvant CFA/IFA isreported in WO 9927944. The use of compositions comprising Aβ fragmentslinked to polypeptides such as diphtheria toxin for promoting an immuneresponse against Aβ is also disclosed in WO 99/27944. However, no dataof immunization are provided.

A monoclonal antibody recognizing an epitope within the N-terminus(1-16) of Aβ (antibody 6C6) has been shown to protect PC12 cells fromneurotoxicity of fibrillar β-amyloid, and to disaggregate β-amyloid invitro (Solomon B. et al., Proc. Natl. Acad. Sci. USA (1997)). Amonoclonal antibody raised against Aβ1-28, was also shown to suppressβ-amyloid aggregation in vitro (Solomon B. et al., Proc. Natl. Acad.Sci. USA (1996)). Frenkel et al., (J. Neuroimmunol. 88: 85-90 (1998))have later identified the epitope of two anti-aggregating antibodies,10D5 and 6C6, as being the epitope “EFRH”, i.e. Aβ3-6. In contrast, anantibody specific for Aβ1-7 was unable to prevent (1-amyloid aggregation(Frenkel D. et al., J. Neuroimmunol. 95: 136-142 (1999)).

Aβ1-42 is fibrillogenic, and indeed, the vaccine composition describedin WO 99/27944 used Aβ1-42 treated in such a way that it can formaggregates. It has been shown that those fibrils are toxic for neuronalcell cultures (Yankner et al., Science 245: 417-420 (1989)), and that atoxic effect is also observed when injected into animal brains(Sigurdson et al., Neurobiol. Aging 17: 893-901 (1996); Sigurdson etal., Neurobiol. Aging 18: 591-608 (1997)). Walsh et al., (Nature416:535-539 (2002)) report that natural oligomers of Aβ are formedwithin intracellular vesicles. Those oligomers inhibited long termpotentiation in rats in vivo and disrupted synaptic plasticity atconcentrations found in human brain and cerebrospinal fluid.

In another study, Bard, F. et al. (Nature Medicine 6:916-19 (2000))reported that peripheral administration of antibodies raised againstAβ₁₋₄₂, was able to reduce amyloid burden, despite relatively modestserum levels. This study utilized either polyclonal antibodies raisedagainst Aβ₁₋₄₂, or monoclonal antibodies raised against syntheticfragments derived from different regions of Aβ. Thus induction ofantibodies against Aβ peptides bears promises as a potential therapeutictreatment for Alzheimer disease.

Mucosal administration of an antigen associated with β-amyloid plaques,such as β-amyloid peptide and Aβ1-40, has been described in WO99/27949.Mucosal administration is said to suppress certain cytokine responsesassociated with Alzheimer's disease, and to enhance certain othercytokine responses associated with the suppression of inflammatoryresponses linked to the disease. It is thought that suppression of theinflammatory responses is effected by the “elicitation of T-cellscharacterized by an anti-inflammatory cytokine profile”. Suitableantigens, as described in WO9927949, include antigens specific for AD,and which are recognized by immune T-cells of a human or animal host.

Fusion of epitopes of a monoclonal antibody recognizing Aβ to coatproteins of filamentous phages is described in WO 01/18169. Immunizationof mice with the filamentous phages displaying the 15-mer epitopeVHEPHEFRHVALNPV (SEQ ID NO: 89) on the coat protein VIII resulted inantibodies recognizing Aβ1-16, and Aβ1-40. This was demonstrated in aninhibition ELISA using Aβ peptides, and an IC₅₀ of 1 μM was found forinhibition of the binding of the sera to Aβ1-16 with Aβ1-40. Solomon (WO01118169), however, provides no indication that the sera elicitedagainst the filamentous phages carrying the VHEPIHEFRHVALNPV epitope(SEQ ID) NO: 89), bind to amyloid plaques or neuritic plaques of AD.

There are a number of drawbacks in using sequences differing from theantigen against which an immune response is to be elicited forimmunization. First, antibodies against part of the sequence foreign tothe antigen or antigenic determinant may be induced. Second, theconformation of the antigen in the context of the foreign flankingsequence element may be different than in the context of the full-lengthantigen. Thus, although antibodies cross-reacting to the antigen may beelicited, their binding to the antigen may be suboptimal. The finespecificity of those elicited antibody may also not correspond to thespecificity of antibodies elicited against the antigen itself, asadditional sid-chains different from the residues present on thefull-length Aβ are present in the epitope. Finally, a 15-mer amino-acidsequence may contain T-cell epitopes. Display of the epitope YYEFRH (SEQID NO: 90) on the protein III of filamentous phage coat, of which 3-5copies only are usually present on each phage, is also disclosed in WO01/18169. Several problems arise when using infectious phages as carrierfor immunization. First, production of infectious agents in large scaleand in sufficient quantity for large immunization campaigns isproblematic. Second, the presence of the DNA of the phage containingantibiotic resistance genes in the vaccine is not unproblematic and is asafety issue. Finally, the feasibility and efficacy of irradiation oflarge quantities of phages, in the case where non-infectious phages areused as vaccine, is unresolved.

Aβ analogues, wherein Aβ is modified to include T helper epitopes havebeen described (WO 01/62284). Immunization of TgRND8+ mice, transgenicfor human APP, with the Aβ analogue resulted in a 4- to 7.5-fold higherantibody titer over immunization with Aβ1-42 in the absence of adjuvant.

Recent studies demonstrated that a vaccination-induced reduction inbrain amyloid deposits has the potential to improve cognitive functions(Schenk, D., et al. Nature 400: 173-177 (1999); Janus, C. et al., Nature408: 979-982 (2000); Morgan, D. et al., Nature 408: 982-985 (2000)).

The autopsy of a patient immunised with aggregated Aβ1-42 in theAdjuvant QS21 has revealed the presence of a T-lymphocytemeningoencephalitis and infiltration of cerebral white matter bymacrophages (Nicoll, J. A. et al., Nature Med. 9: 448-452 (2003)).

Recently, a publication has reported 18 cases of meningoencephalitis inpatients immunized by the AN1792, a vaccine composed of aggregatedAβ1-42 and QS-21 as adjuvant (Orgogozo J.-M. et al., Neurology 61: 46-54(2003)). T-cell activation is reported as a potential mechanismresponsible for the disease, while there was no clear relation betweendisease and anti-Aβ1-42 titers in the serum.

It is well established that the administration of purified proteinsalone is usually not sufficient to elicit a strong immune response;isolated antigen generally must be given together with helper substancescalled adjuvants. Within these adjuvants, the administered antigen isprotected against rapid degradation, and the adjuvant provides anextended release of a low level of antigen. In the present invention, Aβpeptides are made immunogenic through binding to a VLP and do notrequire an adjuvant.

One way to improve the efficiency of vaccination is thus to increase thedegree of repetitiveness of the antigen applied. Unlike isolatedproteins, viruses induce prompt and efficient immune responses in theabsence of any adjuvants both with and without T-cell help (Bachmann andZinkernagel, Ann. Rev. Immunol: 15:235-270 (1991)). Although virusesoften consist of few proteins, they are able to trigger much strongerimmune responses than their isolated components. For B-cell responses,it is known that one crucial factor for the immunogenicity of viruses isthe repetitiveness and order of surface epitopes. Many viruses exhibit aquasi-crystalline surface that displays a regular array of epitopeswhich efficiently crosslinks epitope-specific immunoglobulins on B cells(Bachmann and Zinkernagel, Immunol. Today 17:553-558 (1996)). Thiscrosslinking of surface immunoglobulins on B cells is a strongactivation signal that directly induces cell-cycle progression and theproduction of IgM antibodies. Further, such triggered B cells are ableto activate T helper cells, which in turn induce a switch from IgM toIgG antibody production in B cells and the generation of long-lived Bcell memory—the goal of any vaccination (Bachmann and Zinkernagel, Ann.Rev. Immunol. 15:235-270 (1997)). Viral structure is even linked to thegeneration of anti-antibodies in autoimmune disease and as a part of thenatural response to pathogens (see Fehr, T., et al., J Exp. Med.185:1785-1792 (1997)). Thus, antibodies presented by a highly organizedviral surface are able to induce strong anti-antibody responses.

As indicated, however, the immune system usually fails to produceantibodies against self-derived structures. For soluble antigens presentat low concentrations, this is due to tolerance at the Th cell level.Under these conditions, coupling the self-antigen to a carrier that candeliver T help may break tolerance. For soluble proteins present at highconcentrations or membrane proteins at low concentration, B and Th cellsmay be tolerant. However, B cell tolerance may be reversible (anergy)and can be broken by administration of the antigen in a highly organizedfashion coupled to a foreign carrier (Bachmann and Zinkernagel, Ann.Rev. Immunol. 15:235-270 (1997)). As shown in pending U.S. applicationSer. No. 10/050,902 filed on Jan. 18, 2002, strong immune responsescould be induced with compositions comprising Aβ peptides (Aβ1-15,Aβ1-27 and Aβ33-42, which is a self-antigen in mice) bound to a VLP. Inparticular, the above-mentioned human Aβ peptides bound to the VLP ofRNA phage Qβ induced high Aβ specific titers in human APP transgenicmice (described in Example) demonstrating that tolerance to theself-antigen Aβ could be overcome by immunizing with Aβ peptides boundto a VLP.

There is thus a need for highly immunogenic safe compositions andvaccines, respectively, to treat Alzheimer diseases, in particular,using immunogens devoid of T-cell epitopes and adjuvants, respectively,which might elicit side-effects, and still being capable of inducinghigh antibody titers, which antibodies, furthermore, being capable ofbinding to amyloid plaques.

BRIEF SUMMARY OF THE INVENTION

We have now found that Aβ1-6 peptide, which is bound to a core particlehaving a structure with an inherent repetitive organization, and herebyin particular to virus-like-particles (VLPs) and subunits of VLPs,respectively, leading to highly ordered and repetitive conjugatesrepresent a potent immunogen for the induction of antibodies specificfor Aβ1-6. Therefore, the present invention provides a prophylactic andtherapeutic mean for the treatment of Alzheimer's disease, which isbased on an ordered and repetitive Aβ1-6-core particle array, and inparticular on a VLP-Aβ1-6 peptide conjugate and -array, respectively.This prophylactic and therapeutic is able to induce high titers ofanti-Aβ1-6 peptide antibodies, which are cross-reactive to soluble Aβand are capable of binding to human amyloid plaques of a human APPtransgenic mouse model and to AD amyloid plaques. Furthermore, theelicited antibodies do not bind to APP on brain sections.

Moreover, the present invention provides for new compositions, vaccinesand methods of treatment of AD. The compositions and vaccines comprisingAβ 1-6 peptides are devoid of T-cell epitopes and induce antibodiesbinding AD plaques and soluble Aβ. The Aβ 1-6 peptides are presented tothe immune system of the patient in a highly repetitive and orderedfashion through binding of the Aβ peptides or to a core particle,preferably to a VLP, and even more preferably to a VLP of a RNA phage.

In a preferred embodiment, the antigen or antigenic determinant is thehuman amyloid beta peptide Aβ1-6 (DAEFRH; SEQ ID NO: 75) being afragment of Aβ (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGOVVIA (SEQ ID NO:91), wherein the human amyloid beta peptide Aβ1-6 is bound to the coreparticle and VLP, respectively. The amyloid beta protein is provided inSEQ ID NO: 92. The amyloid beta precursor protein is provided in SEQ IDNO: 93.

The present invention, thus, provides for a composition comprising: (a)a core particle with at least one first attachment site; and (b) atleast one antigen or antigenic determinant with at least one secondattachment site, wherein said antigen or antigenic determinant is aAβ1-6 peptide, and wherein said second attachment site being selectedfrom the group consisting of (i) an attachment site not naturallyoccurring with said antigen or antigenic determinant; and (ii) anattachment site naturally occurring with said antigen or antigenicdeterminant, wherein said second attachment site is capable ofassociation to said first attachment site; and wherein said antigen orantigenic determinant and said core particle interact through saidassociation to form an ordered and repetitive antigen array. Preferredembodiments of core particles suitable for use in the present inventionare a virus, a virus-like particle, a bacteriophage, a virus-likeparticle of a RNA-phage, a bacterial pilus or flagella or any other coreparticle having an inherent repetitive structure capable of forming anordered and repetitive antigen array in accordance with the presentinvention.

The Aβ fragments of the present invention are soluble and generally donot form aggregates. Moreover, they are bound, and preferably covalentlybound to a core particle and VLP, respectively. Therefore, thecompositions of the invention do not bear the risk of inducing toxiceffects such as seeding of amyloid deposition.

It is an unexpected finding of this invention that a high titer ofantibodies cross-reactive with soluble Aβ1 and AD amyloid plaques couldbe obtained with a composition comprising the Aβ1-6 peptide bound to a acore particle and VLP, respectively. In particular. VLP have been shownto mediate induction of antibodies against self antigens, thus breakingself-tolerance (WO 02/056905, the disclosure of which is herewithincorporated by reference in its entirety). Furthermore, the small sizeof this epitope precludes the presence of T-cell epitopes, thusproviding new compositions that do not induce Aβ specific T-cellresponses. In addition, the elicited antibodies do not bind to APP onbrain sections. Thus, the present invention provides for a safe vaccinecomposition for the prevention and treatment of AD.

More specifically, the invention provides a composition comprising anordered and repetitive antigen or antigenic determinant array, andhereby in particular Aβ1-6 peptide VLP conjugates. More specifically,the invention provides a composition comprising a virus-like particleand at least one Aβ1-6 peptide bound thereto. The invention alsoprovides a process for producing the conjugates and the ordered andrepetitive arrays, respectively. The compositions of the invention areuseful in the production of vaccines for the treatment of Alzheimer'sdisease and as a pharmaccine to prevent or cure Alzheimer's disease andto efficiently induce immune responses, in particular antibodyresponses. Furthermore, the compositions of the invention areparticularly useful to efficiently induce self-specific immune responseswithin the indicated context.

In the present invention, a Aβ1-6 peptide is bound to a core particleand VLP, respectively, typically in an oriented manner, yielding anordered and repetitive Aβ1-6 peptide antigen array. Furthermore, thehighly repetitive and organized structure of the core particles andVLPs, respectively, mediates the display of the Aβ peptide in a highlyordered and repetitive fashion leading to a highly organized andrepetitive antigen array. Furthermore, binding of the Aβ1-6 peptide tothe core particle and VLP, respectively, provides T helper cellepitopes, since the core particle and VLP is foreign to the hostimmunized with the core particle-Aβ1-6 peptide array and VLP-Aβ1-6peptide array, respectively. Those arrays differ from prior artconjugates, in particular, in their highly organized structure,dimensions, and in the repetitiveness of the antigen on the surface ofthe array.

In one aspect of the invention, the Aβ1-6 peptide is chemicallysynthesized, while the core particle and the VLP, respectively, isexpressed and purified from an expression host suitable for the foldingand assembly of the core particle and the VLP, respectively. The Aβ1-6peptide array is then assembled by binding the Aβ1-6 peptide to the coreparticle and the VLP, respectively.

In another aspect, the present invention provides for a compositioncomprising (a) a virus-like particle, and (b) at least one antigen orantigenic determinant, wherein said antigen or said antigenicdeterminant is an Aβ1-6 peptide, and wherein said at least one antigenor antigenic determinant is bound to said virus-like particle.

In a further aspect, the present invention provides for a pharmaceuticalcomposition comprising (a) the inventive composition, and (b) anacceptable pharmaceutical carrier.

In still a further aspect, the present invention provides for a vaccinecomposition comprising a composition, wherein said compositioncomprising (a) a virus-like particle; and (b) at least one antigen orantigenic determinant, wherein said antigen or said antigenicdeterminant is a Aβ1-6 peptide; and wherein said at least one antigen orantigenic determinant is bound to said virus-like particle.

In another aspect, the present invention provides for a method ofimmunization comprising administering the inventive composition, theinventive pharmaceutical composition or the inventive vaccine to ananimal.

In still a further aspect, the present invention provides for a processfor producing an inventive composition comprising (a) providing avirus-like particle; and (b) providing at least one antigen or antigenicdeterminant, wherein said antigen or said antigenic determinant is aAβ1-6 peptide; (c) combining said virus-like particle and said at leastone antigen or antigenic determinant so that said at least one antigenor antigenic determinant is bound to said virus-like particle.

Analogously, the present invention provides a process for producing acomposition of claim 1 comprising: (a) providing a core particle with atleast one first attachment site; (b) providing at least one antigen orantigenic determinant with at least one second attachment site, whereinsaid antigen or antigenic determinant is a Aβ1-6 peptide, and whereinsaid second attachment site being selected from the group consisting of(i) an attachment site not naturally occurring with said antigen orantigenic determinant; and (ii) an attachment site naturally occurringwith said antigen or antigenic determinant; and wherein said secondattachment site is capable of association to said first attachment site;and (c) combining said core particle and said at least one antigen orantigenic determinant, wherein said antigen or antigenic determinant andsaid core particle interact through said association to form an orderedand repetitive antigen array.

In a further aspect, the present invention provides for a use of acomposition of claim 1 for the manufacture of a medicament for treatmentof Alzheimer's disease.

In a still further aspect, the present invention provides for a use of acomposition of claim 1 for the preparation of a medicament for thetherapeutic or prophylactic treatment of Alzheimer's disease.Furthermore, in a still further aspect, the present invention providesfor a use of a composition of claim 1, either in isolation or incombination with other agents, or with explicit absence of specificsubstances such as adjuvants, for the manufacture of a composition,pharmaceutical composition, vaccine, drug or medicament for therapy orprophylaxis of Alzheimer's disease, and/or for stimulating the mammalianimmune system.

Therefore, the invention provides, in particular, vaccine compositionswhich are suitable for preventing and/or attenuating Alzheimer's diseaseor conditions related thereto. The invention further providesimmunization and vaccination methods, respectively, for preventingand/or attenuating Alzheimer's disease or conditions related thereto inhumans. The inventive compositions may be used prophylactically ortherapeutically.

In specific embodiments, the invention provides methods for preventingand/or attenuating Alzheimer's disease or conditions related theretowhich are caused or exacerbated by “self” gene products, i.e. “selfantigens” as used herein. In related embodiments, the invention providesmethods for inducing immunological responses in animals and individuals,respectively, which lead to the production of antibodies that preventand/or attenuate Alzheimer's disease or conditions related thereto,which are caused or exacerbated by “self” gene products.

As would be understood by one of ordinary skill in the art, whencompositions of the invention are administered to an animal or a human,they may be in a composition which contains salts, buffers, adjuvants,or other substances which are desirable for improving the efficacy ofthe composition. Examples of materials suitable for use in preparingpharmaceutical compositions are provided in numerous sources includingRemington's Pharmaceutical Sciences (Osol, A, ed., Mack Publishing Co.(1990)).

Compositions of the invention are said to be “pharmacologicallyaccept-able” if their administration can be tolerated by a recipientindividual. Further, the compositions of the invention will beadministered in a “therapeutically effective amount” (i.e., an amountthat produces a desired physiological effect).

The compositions of the present invention may be administered by variousmethods known in the art, but will normally be administered byinjection, infusion, inhalation, oral administration, or other suitablephysical methods. The compositions may alternatively be administeredintramuscularly, intravenously, or subcutaneously. Components ofcompositions for administration include sterile aqueous (e.g.physiological saline) or non-aqueous solutions and suspensions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Carriers or occlusive dressings can be used to increaseskin permeability and enhance antigen absorption.

Other embodiments of the present invention will be apparent to one ofordinary skill in light of what is known in the art, the followingdrawings and description of the invention, and the claims.

BRIEF DESCRIPTION OF VIEWS OF THE FIGURES

FIG. 1 depicts the SDS-PAGE gel, run under reducing conditions, showingthe result of the coupling of the Aβ1-6 peptide (NH2-DAEFRHGGC-CONH2)(SEQ ID NO: 77) to the VLP of Qβ coat protein.

FIG. 2 shows the ELISA analysis of the antibodies specific for Aβ1-6 insera of mice immunized with Aβ1-6 peptide coupled to the VLP of Qβ coatprotein.

FIG. 3 shows the ELISA analysis of the antibodies specific for Aβ1-40 insera of mice immunized with Aβ1-6 peptide coupled to the VLP of Qβ coatprotein.

FIG. 4 A-B show a brain section of an APP23 mouse (A) and an entorhinalcortex section from an AD patient (B) stained with sera of miceimmunized with Aβ1-6 peptide coupled to the VLP of Qβ coat protein.

FIG. 5 A-E show brain sections of an APP23 mouse stained with sera ofmice immunized with Aβ1-6 peptide coupled to the VLP of Qβ coat protein,or with a polyclonal rabbit antiserum specific for the C-terminus ofhuman or mouse APP.

FIG. 6 shows the result of the immunization of rhesus monkeys with humanAβ1-6 coupled to Qβ VLP as measured in an ELISA assay.

FIG. 7 A-B shows the result of the binding to plaques of sera frommonkeys immunized with human Aβ1-6 coupled to Qβ VLP, as measured byhistology on human AD and transgenic mouse plaques.

FIG. 8 depicts the SDS-PAGE analysis of the coupling of murine Aβ1-6 toAP205 VLP.

FIG. 9 shows the result of the immunisation of mice with murine Aβ1-6coupled to AP205 as measured in an ELISA assay.

FIG. 10 shows the analysis by ELISA of the anti-Aβ40 and anti-Aβ42titers in the sera of “Swedish/London” transgenic mice immunized withQβhAβ1-6 between 9.5 and 19 months of age.

FIG. 11A-B shows the immunohistochemical staining of brain sections of“Swedish/London” transgenic mice immunized with QβhβA1-6 or PBS.

FIG. 12 A-H shows the quantification of plaque deposition in“Swedish/London” transgenic mice immunized with QβhAβ1-6, Q or PBSbetween 9.5 and 19 months of age.

FIG. 13 A-H shows the quantification of plaque deposition in“Swedish/London” transgenic mice immunized with QβhAβ1-6 or PBS between13.5 and 19 months of age.

FIG. 14 shows the analysis by ELISA of the anti-Aβ40 and anti-Aβ42titers in the sera of “Swedish” transgenic mice immunized with QβhAβ1-6.

FIG. 15 A-B shows the immunohistochemical staining of brain sectionsfrom “Swedish” transgenic mice immunized with QβhAβ1-6 or PBS.

FIG. 16 A-B shows the quantification of plaque deposition in “Swedish”transgenic mice immunized with QβhAβ1-6 or PBS.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are hereinafter described.

1. DEFINITIONS

Aβ1-6 peptide: An Aβ1-6 peptide as used herein refers to peptides havinga sequence corresponding to the human Aβ1-6 sequence, or homologous tothe human Aβ1-6 sequence. Sequences homologous to the human Aβ1-6sequence include, but are not limited to the Aβ1-6 sequences of otherspecies and hereby including, but not limited to, the sequence ofprimate, rabbit, guinea pig, Xenopus Laevis, frog, mouse and rat Aβ1-6.The Aβ1-6 sequences from Xenopus Laevis or frog, although differing fromhuman Aβ1-6 at two positions, have conservative mutations (Ala-Ser,Phe-Tyr), and are still considered to be homologous to Aβ1-6 inaccordance with this definition. In accordance with the presentinvention, however, the Aβ1-6 peptide is typically modified, such that asecond attachment site is attached thereto. Preferably, the secondattachment site is modified with a linker or an amino acid linkercomprising a second attachment site for binding to a core particle andVLP, respectively. While referring herein to Aβ1-6 peptides, a modifiedAβ1-6 peptide, as indicated above, i.e. Aβ1-6 peptides with a secondattachment site attached thereto, shall be encompassed. Typically,however, the modifications are explicitly indicated in thespecification. Further preferred embodiments of an Aβ1-6 peptide beingan antigen or antigenic determinant in accordance with the presentinvention become apparent as this specification proceeds.

Adjuvant: The term “adjuvant” as used herein refers to non-specificstimulators of the immune response or substances that allow generationof a depot in the host which when combined with the vaccine andpharmaceutical composition, respectively, of the present invention mayprovide for an even more enhanced immune response. A variety ofadjuvants can be used. Examples include complete and incomplete Freund'sadjuvant, aluminum hydroxide and modified muramyldipeptide. Furtheradjuvants are mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and Corynebacterium parvum. Such adjuvants are also well known in theart. Further adjuvants that can be administered with the compositions ofthe invention include, but are not limited to, Monophosphoryl lipidimmunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts(Alum), MF-59, OM-174, OM-197, OM-294, and Virosomal adjuvanttechnology. The adjuvants can also comprise a mixture of thesesubstances.

Immunologically active saponin fractions having adjuvant activityderived from the bark of the South American tree Quillaja SaponariaMolina are known in the art. For example QS21, also known as QA21, is anHplc purified fraction from the Quillaja Saponaria Molina tree and it'smethod of its production is disclosed (as QA21) in U.S. Pat. No.5,057,540. Quillaja saponin has also been disclosed as an adjuvant byScott et al. Int. Archs. Allergy Appl. Immun., 1985, 77, 409.Monosphoryl lipid A and derivatives thereof are known in the art. Apreferred derivative is 3 de-o-acylated monophosphoryl lipid A, and isknown from British Patent No. 2220211. Further preferred adjuvants aredescribed in WO00/00462, the disclosure of which is herein incorporatedby reference.

However, an advantageous feature of the present invention is the highimmunogenicity of the inventive compositions. As already outlined hereinor will become apparent as this specification proceeds, vaccines andpharmaceutical compositions devoid of adjuvants are provided, in furtheralternative or preferred embodiments, leading to vaccines andpharmaceutical compositions for treating AD being devoid of adjuvantsand, thus, having a superior safety profile since adjuvants may causeside-effects. The term “devoid” as used herein in the context ofvaccines and pharmaceutical compositions for treating AD refers tovaccines and pharmaceutical compositions that are used withoutadjuvants.

Amino acid linker: An “amino acid linker”, or also just termed “linker”within this specification, as used herein, either associates the antigenor antigenic determinant with the second attachment site, or morepreferably, already comprises or contains the second attachment site,typically—but not necessarily—as one amino acid residue, preferably as acysteine residue. The term “amino acid linker” as used herein, however,does not intend to imply that such an amino acid linker consistsexclusively of amino acid residues, even if an amino acid linkerconsisting of amino acid residues is a preferred embodiment of thepresent invention. The amino acid residues of the amino acid linker are,preferably, composed of naturally occurring amino acids or unnaturalamino acids known in the art, all-L or all-D or mixtures thereof.However, an amino acid linker comprising a molecule with a sulfhydrylgroup or cysteine residue is also encompassed within the invention. Sucha molecule comprise preferably a C1-C6 alkyl-, cycloalkyl (C5,C6), arylor heteroaryl moiety. However, in addition to an amino acid linker, alinker comprising preferably a C1-C6 alkyl-, cycloalkyl-(C5,C6), aryl-or heteroaryl-moiety and devoid of any amino acid(s) shall also beencompassed within the scope of the invention. Association between theantigen or antigenic determinant or optionally the second attachmentsite and the amino acid linker is preferably by way of at least onecovalent bond, more preferably by way of at least one peptide bond.

Animal: As used herein, the term “animal” is meant to include, forexample, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys,horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken,reptiles, fish, insects and arachnids.

Antibody: As used herein, the term “antibody” refers to molecules whichare capable of binding an epitope or antigenic determinant. The term ismeant to include whole antibodies and antigen-binding fragments thereof,including single-chain antibodies. Most preferably the antibodies arehuman antigen binding antibody fragments and include, but are notlimited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv),single-chain antibodies, disulfide-linked Fvs (sdFv) and fragmentscomprising either a V_(L) or V_(H) domain. The antibodies can be fromany animal origin including birds and mammals. Preferably, theantibodies are human, murine, rabbit, goat, guinea pig, camel, horse orchicken. As used herein, “human” antibodies include antibodies havingthe amino acid sequence of a human immunoglobulin and include antibodiesisolated from human immunoglobulin libraries or from animals transgenicfor one or more human immunoglobulins and that do not express endogenousimmunoglobulins, as described, for example, in U.S. Pat. No. 5,939,598by Kucherlapati et al.

Antigen: As used herein, the term “antigen” refers to a molecule capableof being bound by an antibody or a T cell receptor (TCR) if presented byMHC molecules. The term “antigen”, as used herein, also encompassesT-cell epitopes. An antigen is additionally capable of being recognizedby the immune system and/or being capable of inducing a humoral immuneresponse and/or cellular immune response leading to the activation of B-and/or T-lymphocytes. This may, however, require that, at least incertain cases, the antigen contains or is linked to a Th cell epitopeand is given in adjuvant. An antigen can have one or more epitopes (B-and T-epitopes). The specific reaction referred to above is meant toindicate that the antigen will preferably react, typically in a highlyselective manner, with its corresponding antibody or TCR and not withthe multitude of other antibodies or TCRs which may be evoked by otherantigens. Antigens as used herein may also be mixtures of severalindividual antigens.

Antigenic determinant: As used herein, the term “antigenic determinant”is meant to refer to that portion of an antigen that is specificallyrecognized by either B- or T-lymphocytes. B-lymphocytes responding toantigenic determinants produce antibodies, whereas T-lymphocytes respondto antigenic determinants by proliferation and establishment of effectorfunctions critical for the mediation of cellular and/or humoralimmunity.

Association: As used herein, the term “association” as it applies to thefirst and second attachment sites, refers to the binding of the firstand second attachment sites that is preferably by way of at least onenon-peptide bond. The nature of the association may be covalent, ionic,hydrophobic, polar or any combination thereof, preferably the nature ofthe association is covalent.

Attachment Site, First: As used herein, the phrase “first attachmentsite” refers to an element of non-natural or natural origin, to whichthe second attachment site located on the antigen or antigenicdeterminant may associate. The first attachment site may be a protein, apolypeptide, an amino acid, a peptide, a sugar, a polynucleotide, anatural or synthetic polymer, a secondary metabolite or compound(biotin, fluorescein, retinol, digoxigenin, metal ions,phenylmethylsulfonylfluoride), or a combination thereof, or a chemicallyreactive group thereof. The first attachment site is located, typicallyand preferably on the surface, of the core particle such as, preferablythe virus-like particle. Multiple first attachment sites are present onthe surface of the core and virus-like particle, respectively, typicallyin a repetitive configuration.

Attachment Site, Second: As used herein, the phrase “second attachmentsite” refers to an element associated with the antigen or antigenicdeterminant to which the first attachment site located on the surface ofthe core particle and virus-like particle, respectively, may associate.The second attachment site of the antigen or antigenic determinant maybe a protein, a polypeptide, a peptide, a sugar, a polynucleotide, anatural or synthetic polymer, a secondary metabolite or compound(biotin, fluorescein, retinol, digoxigenin, metal ions,phenylmethylsulfonylfluoride), or a combination thereof, or a chemicallyreactive group thereof. At least one second attachment site is presenton the antigen or antigenic determinant. The term “antigen or antigenicdeterminant with at least one second attachment site” refers, therefore,to an antigen or antigenic construct comprising at least the antigen orantigenic determinant and the second attachment site. However, inparticular for a second attachment site, which is of non-natural origin,i.e. not naturally occurring within the antigen or antigenicdeterminant, these antigen or antigenic constructs comprise an “aminoacid linker”.

Bound: As used herein, the term “bound” refers to binding or attachmentthat may be covalent, e.g., by chemically coupling, or non-covalent,e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc.Covalent bonds can be, for example, ester, ether, phosphoester, amide,peptide, imide, carbon-sulfur bonds, carbon-phosphorus bonds, and thelike. The term “bound” is broader than and includes terms such as“coupled,” “fused” and “attached”.

Coat protein(s): As used herein, the term “coat protein(s)” refers tothe protein(s) of a bacteriophage or a RNA-phage capable of beingincorporated within the capsid assembly of the bacteriophage or theRNA-phage. However, when referring to the specific gene product of thecoat protein gene of RNA-phages the term “CP” is used. For example, thespecific gene product of the coat protein gene of RNA-phage Qβ isreferred to as “Qβ CP”, whereas the “coat proteins” of bacteriophage Qβcomprise the “Qβ CP” as well as the A1 protein. The capsid ofBacteriophage Qβ is composed mainly of the Qβ CP, with a minor contentof the A1 protein. Likewise, the VLP Qβ coat protein contains mainly QβCP, with a minor content of A1 protein.

Core particle: As used herein, the term “core particle” refers to arigid structure with an inherent repetitive organization. A coreparticle as used herein may be the product of a synthetic process or theproduct of a biological process.

Coupled: The term “coupled”, as used herein, refers to attachment bycovalent bonds or by strong non-covalent interactions, typically andpreferably to attachment by covalent bonds. Any method normally used bythose skilled in the art for the coupling of biologically activematerials can be used in the present invention.

Effective Amount: As used herein, the term “effective amount” refers toan amount necessary or sufficient to realize a desired biologic effect.An effective amount of the composition would be the amount that achievesthis selected result, and such an amount could be determined as a matterof routine by a person skilled in the art. For example, an effectiveamount for treating an immune system deficiency could be that amountnecessary to cause activation of the immune system, resulting in thedevelopment of an antigen specific immune response upon exposure toantigen. The term is also synonymous with “sufficient amount.”

The effective amount for any particular application can vary dependingon such factors as the disease or condition being treated, theparticular composition being administered, the size of the subject,and/or the severity of the disease or condition. One of ordinary skillin the art can empirically determine the effective amount of aparticular composition of the present invention without necessitatingundue experimentation.

Epitope: As used herein, the term “epitope” refers to continuous ordiscontinuous portions of a polypeptide having antigenic or immunogenicactivity in an animal, preferably a mammal, and most preferably in ahuman. An epitope is recognized by an antibody or a T cell through its Tcell receptor in the context of an MHC molecule. An “immunogenicepitope,” as used herein, is defined as a portion of a polypeptide thatelicits an antibody response or induces a T-cell response in an animal,as determined by any method known in the art. (See, for example, Geysenet al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term“antigenic epitope,” as used herein, is defined as a portion of aprotein to which an antibody can immunospecifically bind its antigen asdetermined by any method well known in the art. Immunospecific bindingexcludes non-specific binding but does not necessarily excludecross-reactivity with other antigens. Antigenic epitopes need notnecessarily be immunogenic. Antigenic epitopes can also be T-cellepitopes, in which case they can be bound immunospecifically by a T-cellreceptor within the context of an MHC molecule.

An epitope can comprise 3 amino acids in a spatial conformation which isunique to the epitope. Generally, an epitope consists of at least about5 such amino acids, and more usually, consists of at least about 8-10such amino acids. If the epitope is an organic molecule, it may be assmall as Nitrophenyl.

Fusion: As used herein, the term “fusion” refers to the combination ofamino acid sequences of different origin in one polypeptide chain byin-frame combination of their coding nucleotide sequences. The term“fusion” explicitly encompasses internal fusions, i.e., insertion ofsequences of different origin within a polypeptide chain, in addition tofusion to one of its termini.

Immune response: As used herein, the term “immune response” refers to ahumoral immune response and/or cellular immune response leading to theactivation or proliferation of B- and/or T-lymphocytes and/or andantigen presenting cells. In some instances, however, the immuneresponses may be of low intensity and become detectable only when usingat least one substance in accordance with the invention. “Immunogenic”refers to an agent used to stimulate the immune system of a livingorganism, so that one or more functions of the immune system areincreased and directed towards the immunogenic agent. An “immunogenicpolypeptide” is a polypeptide that elicits a cellular and/or humoralimmune response, whether alone or linked to a carrier in the presence orabsence of an adjuvant. Preferably, antigen presenting cell may beactivated.

A substance which “enhances” an immune response refers to a substance inwhich an immune response is observed that is greater or intensified ordeviated in any way with the addition of the substance when compared tothe same immune response measured without the addition of the substance.For example, the lytic activity of cytotoxic T cells can be measured,e.g. using a ⁵¹Cr release assay, in samples obtained with and withoutthe use of the substance during immunization. The amount of thesubstance at which the CTL lytic activity is enhanced as compared to theCTL lytic activity without the substance is said to be an amountsufficient to enhance the immune response of the animal to the antigen.In a preferred embodiment, the immune response in enhanced by a factorof at least about 2, more preferably by a factor of about 3 or more. Theamount or type of cytokines secreted may also be altered. Alternatively,the amount of antibodies induced or their subclasses may be altered.

Immunization: As used herein, the terms “immunize” or “immunization” orrelated terms refer to conferring the ability to mount a substantialimmune response (comprising antibodies and/or cellular immunity such aseffector CTL) against a target antigen or epitope. These terms do notrequire that complete immunity be created, but rather that an immuneresponse be produced which is substantially greater than baseline. Forexample, a mammal may be considered to be immunized against a targetantigen if the cellular and/or humoral immune response to the targetantigen occurs following the application of methods of the invention.

Natural origin: As used herein, the term “natural origin” means that thewhole or parts thereof are not synthetic and exist or are produced innature.

Non-natural: As used herein, the term generally means not from nature,more specifically, the term means from the hand of man.

Non-natural origin: As used herein, the term “non-natural origin”generally means synthetic or not from nature; more specifically, theterm means from the hand of man.

Ordered and repetitive antigen or antigenic determinant array: As usedherein, the term “ordered and repetitive antigen or antigenicdeterminant array” generally refers to a repeating pattern of antigen orantigenic determinant, characterized by a typically and preferablyuniform spacial arrangement of the antigens or antigenic determinantswith respect to the core particle and virus-like particle, respectively.In one embodiment of the invention, the repeating pattern may be ageometric pattern. Typical and preferred examples of suitable orderedand repetitive antigen or antigenic determinant arrays are those whichpossess strictly repetitive paracrystalline orders of antigens orantigenic determinants, preferably with spacings of 0.5 to 30nanometers, more preferably 5 to 15 nanometers.

Pili: As used herein, the term “pili” (singular being “pilus”) refers toextracellular structures of bacterial cells composed of protein monomers(e.g., pilin monomers) which are organized into ordered and repetitivepatterns.

Further, pili are structures which are involved in processes such as theattachment of bacterial cells to host cell surface receptors,inter-cellular genetic exchanges, and cell-cell recognition. Examples ofpill include Type-1 pili, P-pili, FIC pill, S-pili, and 987P-pili.Additional examples of pili are set out below.

Pilus-like structure: As used herein, the phrase “pilus-like structure”refers to structures having characteristics similar to that of pili andcomposed of protein monomers. One example of a “pilus-like structure” isa structure formed by a bacterial cell which expresses modified pilinproteins that do not form ordered and repetitive arrays that areidentical to those of natural pill.

Polypeptide: As used herein, the term “polypeptide” refers to a moleculecomposed of monomers (amino acids) linearly linked by amide bonds (alsoknown as peptide bonds). It indicates a molecular chain of amino acidsand does not refer to a specific length of the product. Thus, peptides,dipeptides, tripeptides, oligopeptides and proteins are included withinthe definition of polypeptide. This term is also intended to refer topost-expression modifications of the polypeptide, for example,glycosolations, acetylations, phosphorylations, and the like. Arecombinant or derived polypeptide is not necessarily translated from adesignated nucleic acid sequence. It may also be generated in anymanner, including chemical synthesis.

Residue: As used herein, the term “residue” is meant to mean a specificamino acid in a polypeptide backbone or side chain.

Self antigen: As used herein, the term “self antigen” refers to proteinsencoded by the host's DNA and products generated by proteins or RNAencoded by the host's DNA are defined as self. In addition, proteinsthat result from a combination of two or several self-molecules or thatrepresent a fraction of a self-molecule and proteins that have a highhomology two self-molecules as defined above (>95%, preferably >97%,more preferably >99%) may also be considered self.

Treatment: As used herein, the terms “treatment”, “treat”, “treated” or“treating” refer to prophylaxis and/or therapy. When used with respectto an infectious disease, for example, the term refers to a prophylactictreatment which increases the resistance of a subject to infection witha pathogen or, in other words, decreases the likelihood that the subjectwill become infected with the pathogen or will show signs of illnessattributable to the infection, as well as a treatment after the subjecthas become infected in order to fight the infection, e.g., reduce oreliminate the infection or prevent it from becoming worse.

Vaccine: As used herein, the term “vaccine” refers to a formulationwhich contains the composition of the present invention and which is ina form that is capable of being administered to an animal. Typically,the vaccine comprises a conventional saline or buffered aqueous solutionmedium in which the composition of the present invention is suspended ordissolved. In this form, the composition of the present invention can beused conveniently to prevent, ameliorate, or otherwise treat acondition. Upon introduction into a host, the vaccine is able to provokean immune response including, but not limited to, the production ofantibodies and/or cytokines and/or the activation of cytotoxic T cells,antigen presenting cells, helper T cells, dendritic cells and/or othercellular responses.

Optionally, the vaccine of the present invention additionally includesan adjuvant which can be present in either a minor or major proportionrelative to the compound of the present invention.

Virus-like particle (VLP): As used herein, the term “virus-likeparticle” refers to a structure resembling a virus particle. Moreover, avirus-like particle in accordance with the invention is non replicativeand noninfectious since it lacks all or part of the viral genome, inparticular the replicative and infectious components of the viralgenome. A virus-like particle in accordance with the invention maycontain nucleic acid distinct from their genome. A typical and preferredembodiment of a virus-like particle in accordance with the presentinvention is a viral capsid such as the viral capsid of thecorresponding virus, bacteriophage, or RNA-phage. The terms “viralcapsid” or “capsid”, as interchangeably used herein, refer to amacromolecular assembly composed of viral protein subunits. Typicallyand preferably, the viral protein subunits assemble into a viral capsidand capsid, respectively, having a structure with an inherent repetitiveorganization, wherein said structure is, typically, spherical ortubular. For example, the capsids of RNA-phages or HBcAg's have aspherical form of icosahedral symmetry. The term “capsid-like structure”as used herein, refers to a macromolecular assembly composed of viralprotein subunits resembling the capsid morphology in the above definedsense but deviating from the typical symmetrical assembly whilemaintaining a sufficient degree of order and repetitiveness.

Virus-like particle of a bacteriophage: As used herein, the term“virus-like particle of a bacteriophage” refers to a virus-like particleresembling the structure of a bacteriophage, being non replicative andnoninfectious, and lacking at least the gene or genes encoding for thereplication machinery of the bacteriophage, and typically also lackingthe gene or genes encoding the protein or proteins responsible for viralattachment to or entry into the host.

This definition should, however, also encompass virus-like particles ofbacteriophages, in which the aforementioned gene or genes are stillpresent but inactive, and, therefore, also leading to non-replicativeand noninfectious virus-like particles of a bacteriophage.

VLP of RNA phage coat protein: The capsid structure formed from theself-assembly of 180 subunits of RNA phage coat protein and optionallycontaining host RNA is referred to as a “VLP of RNA phage coat protein”.A specific example is the VLP of Qβ3 coat protein. In this particularcase, the VLP of Qβ coat protein may either be assembled exclusivelyfrom Qβ CP subunits (generated by expression of a Qβ CP gene containing,for example, a TAA stop codon precluding any expression of the longer A1protein through suppression, see Kozlovska, T. M., et al., Intervirology39: 9-15 (1996)), or additionally contain A1 protein subunits in thecapsid assembly.

Virus particle: The term “virus particle” as used herein refers to themorphological form of a virus. In some virus types it comprises a genomesurrounded by a protein capsid; others have additional structures (e.g.envelopes, tails, etc.).

One, a, or an: When the terms “one.” “a,” or “an” are used in thisdisclosure, they mean “at least one” or “one or more,” unless otherwiseindicated.

As will be clear to those skilled in the art, certain embodiments of theinvention involve the use of recombinant nucleic acid technologies suchas cloning, polymerase chain reaction, the purification of DNA and RNA,the expression of recombinant proteins in prokaryotic and eukaryoticcells, etc. Such methodologies are well known to those skilled in theart and can be conveniently found in published laboratory methodsmanuals (e.g., Sambrook, J. et al., eds., Molecular Cloning. ALaboratory Manual. 2nd. edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al., eds., CurrentProtocols in Molecular Biology, John H. Wiley & Sons, Inc. (1997)).Fundamental laboratory techniques for working with tissue culture celllines (Celis. J., ed., Cell Biology, Academic Press, 2^(nd) edition,(1998)) and antibody-based technologies (Harlow, E. and Lane, D.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1988); Deutscher, M. P., “Guide to ProteinPurification,” Meth. Emymol. 128, Academic Press San Diego (1990);Scopes, R. K., Protein Purificalion Principles and Practice, 3rd ed.,Springer-Verlag, New York (1994)) are also adequately described in theliterature, all of which are incorporated herein by reference.

2. Compositions and Methods for Enhancing an Immune Response

The disclosed invention provides compositions and methods for inducingan immune response against Aβ1-6 peptide in an animal, inducingantibodies capable of binding A1 amyloid plaques and soluble Aβ.Compositions of the invention comprise, or alternatively consist of (a)a core particle with at least one first attachment site; and (b) atleast one antigen or antigenic determinant with at least one secondattachment site, wherein said antigen or antigenic determinant is anAβ1-6 peptide, and wherein said second attachment site being selectedfrom the group consisting of (i) an attachment site not naturallyoccurring with said antigen or antigenic determinant; and (ii) anattachment site naturally occurring with said antigen or antigenicdeterminant, wherein said second attachment site is capable ofassociation to said first attachment site; and wherein said antigen orantigenic determinant and said core particle interact through saidassociation to form an ordered and repetitive antigen array. Morespecifically, compositions of the invention comprise, or alternativelyconsist of, a virus-like particle and at least one antigen or antigenicdeterminant, wherein the antigen or antigenic determinant is a Aβ1-6peptide, and wherein the at least one antigen or antigenic determinantis bound to the virus-like particle so as to form an ordered andrepetitive antigen-VLP-array. Furthermore, the invention convenientlyenables the practitioner to construct such a composition, inter alia,for treatment and/or prophylactic prevention of Alzheimer's disease.Virus-like particles in the context of the present application refer tostructures resembling a virus particle but which are not pathogenic. Ingeneral, virus-like particles lack the viral genome and, therefore, arenoninfectious. Also, virus-like particles can be produced in largequantities by heterologous expression and can be easily purified.

In one embodiment, the core particle comprises, or is selected from agroup consisting of, a virus, a bacterial pilus, a structure formed frombacterial pilin, a bacteriophage, a virus-like particle, a virus-likeparticle of a RNA phage, a viral capsid particle or a recombinant formthereof. Any virus known in the art having an ordered and repetitivecoat and/or core protein structure may be selected as a core particle ofthe invention; examples of suitable viruses include sindbis and otheralphaviruses, rhabdoviruses (e.g. vesicular stomatitis virus),picornaviruses (e.g. human rhino virus. Aichi virus), togaviruses (e.g.,rubella virus), orthomyxoviruses (e.g. Thogoto virus, Balken virus, fowlplague virus), polyomaviruses (e.g., polyomavirus BK, polyomavirus JC,avian polyomavirus BFDV), parvoviruses, rotaviruses, Norwalk virus, footand mouth disease virus, a retrovirus, Hepatitis B virus, Tobacco mosaicvirus, Flock House Virus, and human Papilomavirus, and preferably a RNAphage, bacteriophage Qβ, bacteriophage R17, bacteriophage M11,bacteriophage MX1, bacteriophage NL95, bacteriophage fr, bacteriophageGA, bacteriophage SP, bacteriophage MS2, bacteriophage 12, bacteriophagePP7 (for example, see Table I in Bachmann, M. F. and Zinkernagel, R. M.,Immunol. Today 17:553-558 (1996)).

In a further embodiment, the invention utilizes genetic engineering of avirus to create a fusion between an ordered and repetitive viralenvelope protein and a first attachment site being comprised by, oralternatively or preferably being a heterologous protein, peptide,antigenic determinant or a reactive amino acid residue of choice. Othergenetic manipulations known to those in the art may be included in theconstruction of the inventive compositions; for example, it may bedesirable to restrict the replication ability of the recombinant virusthrough genetic mutation. Furthermore, the virus used for the presentinvention is replication incompetent due to chemical or physicalinactivation or, as indicated, due to lack of a replication competentgenome. The viral protein selected for fusion to the first attachmentsite should have an organized and repetitive structure. Such anorganized and repetitive structure includes paracrystallineorganizations with a spacing of 5-30 nm, preferably 5-15 nm, on thesurface of the virus. The creation of this type of fusion protein willresult in multiple, ordered and repetitive first attachment sites on thesurface of the virus and reflect the normal organization of the nativeviral protein. As will be understood by those in the art, the firstattachment site may be or be a part of any suitable protein,polypeptide, sugar, polynucleotide, peptide (amino acid), natural orsynthetic polymer, a secondary metabolite or combination thereof thatmay serve to specifically attach the antigen or antigenic determinantleading an ordered and repetitive antigen array.

In another embodiment of the invention, the core particle is arecombinant alphavirus, and more specifically, a recombinant Sinbisvirus. Alphaviruses are positive stranded RNA viruses that replicatetheir genomic RNA entirely in the cytoplasm of the infected cell andwithout a DNA intermediate (Strauss, J. and Strauss, E., Microbiol. Rev.58:491-562 (1994)). Several members of the alphavirus family, Sindbis(Xiong, C. et al, Science 243:1188-1191 (1989); Schlesinger, S., TrendsBiotechnol. 11:18-22 (1993)), Semliki Forest Virus (SFV) (Liljeström, P.& Garoff, H., Bio/Technology 9:1356-1361 (1991)) and others (Davis, N.L. et al., Virology 171:189-204 (1989)), have received considerableattention for use as virus-based expression vectors for a variety ofdifferent proteins (Lundstrom, K., Curr. Opin. Biotechnol. 8:578-582(1997); Liljeström, P., Curr. Opin. Biotechnol. 5:495-500 (1994)) and ascandidates for vaccine development. Recently, a number of patents haveissued directed to the use of alphaviruses for the expression ofheterologous proteins and the development of vaccines (see U.S. Pat.Nos. 5,766,602; 5,792,462; 5,739,026; 5,789,245 and 5,814,482). Theconstruction of the alphaviral core particles of the invention may bedone by means generally known in the art of recombinant DNA technology,as described by the aforementioned articles, which are incorporatedherein by reference.

A variety of different recombinant host cells can be utilized to producea viral-based core particle for antigen or antigenic determinantattachment. For example, alphaviruses are known to have a wide hostrange; Sindbis virus infects cultured mammalian, reptilian, andamphibian cells, as well as some insect cells (Clark, H., J. Natl.Cancer Inst. 51:645 (1973); Leake, C., J. Gen. Virol. 35:335 (1977);Stollar, V. in THE TOGAVIRUSIS, R. W. Schlesinger, Ed., Academic Press,(1980), pp. 583-621). Thus, numerous recombinant host cells can be usedin the practice of the invention. BHK, COS, Vero, HeLa and CHO cells areparticularly suitable for the production of heterologous proteinsbecause they have the potential to glycosylate heterologous proteins ina manner similar to human cells (Watson, E. et al, Glycobiology 4:227,(1994)) and can be selected (Zang, M. et al., Bio/Technology 13:389(1995)) or genetically engineered (Renner W. et al., Biotech. Bioeng.4:476 (1995); Lee K. et al. Biotech. Bioeng. 50:336 (1996)) to grow inserum-free medium, as well as in suspension.

Introduction of the polynucleotide vectors into host cells can beeffected by methods described in standard laboratory manuals (see, e.g.,Sambrook, J. et al., eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd.edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989), Chapter 9; Ausubel, F. et al., eds., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997), Chapter 16),including methods such as electroporation, DEAE-dextran mediatedtransfection, transfection, microinjection, cationic lipid-mediatedtransfection, transduction, scrape loading, ballistic introduction, andinfection. Methods for the introduction of exogenous DNA sequences intohost cells are discussed in Felgner, P. et al., U.S. Pat. No. 5,580,859.

Packaged RNA sequences can also be used to infect host cells. Thesepackaged RNA sequences can be introduced to host cells by adding them tothe culture medium. For example, the preparation of non-infectivealpahviral particles is described in a number of sources, including“Sindbis Expression System”, Version C (Invitrogen Catalog No. K750-1).

When mammalian cells are used as recombinant host cells for theproduction of viral-based core particles, these cells will generally begrown in tissue culture. Methods for growing cells in culture are wellknown in the art (see, e.g., Celis, J., ed., CELL BIOLOGY, AcademicPress, 2^(nd) edition, (1998); Sambrook, J. et al., eds., MOLECULARCLONING, A LABORATORY MANUAL, 2nd, edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al.,eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc.(1997); Freshney, R., CULTURE OF ANIMAL CELLS, Alan R. Liss, Inc.(1983)).

Further examples of RNA viruses suitable for use as core particle in thepresent invention include, but are not limited to, the following:members of the family Reoviridae, including the genus Orthoreovirus(multiple serotypes of both mammalian and avian retroviruses), the genusOrbivirus (Bluetongue virus, Eugenangee virus, Kemerovo virus, Africanhorse sickness virus, and Colorado Tick Fever virus), the genusRotavirus (human rotavirus, Nebraska calf diarrhea virus, murinerotavirus, simian rotavirus, bovine or ovine rotavirus, avianrotavirus); the family Picornaviridae, including the genus Enterovirus(poliovirus, Coxsackie virus A and B, enteric cytopathic human orphan(ECHO) viruses, hepatitis A, C, D, E and G viruses, Simianenteroviruses, Murine encephalomyelitis (ME) viruses, Poliovirus muris,Bovine enteroviruses, Porcine enteroviruses, the genus Cardiovirus(Encephalomyocarditis virus (EMC), Mengovirus), the genus Rhinovirus(Human rhinoviruses including at least 113 subtypes; otherrhinoviruses), the genus Apthovirus (Foot and Mouth disease (FMDV); thefamily Calciviridae, including Vesicular exanthema of swine virus, SanMiguel sea lion virus, Feline picornavirus and Norwalk virus; the familyTogaviridae, including the genus Alphavirus (Eastern equine encephalitisvirus, Semliki forest virus, Sindbis virus, Chikungunya virus,O'Nyong-Nyong virus, Ross river virus, Venezuelan equine encephalitisvirus. Western equine encephalitis virus), the genus Flavirius (Mosquitoborne yellow fever virus. Dengue virus, Japanese encephalitis virus. St.Louis encephalitis virus, Murray Valley encephalitis virus, West Nilevirus, Kunjin virus, Central European tick borne virus, Far Eastern tickborne virus, Kyasanur forest virus, Louping III virus, Powassan virus,Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), thegenus Pestivirus (Mucosal disease virus, Hog cholera virus, Borderdisease virus); the family Bunyaviridae, including the genus Bunyvirus(Bunyamwera and related viruses, California encephalitis group viruses),the genus Phiebovirus (Sandfly fever Sicilian virus, Rift Valley fevervirus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus,Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi andrelated viruses); the family Orthomyxoviridae, including the genusInfluenza virus (Influenza virus type A, many human subtypes): Swineinfluenza virus, and Avian and Equine Influenza viruses; influenza typeB (many human subtypes), and influenza type C (possible separate genus);the family paramyxoviridae, including the genus Paramyxovirus(Parainfluenza virus type I, Sendai virus, Hemadsorption virus,Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumpsvirus), the genus Morbillivirus (Measles virus, subacute sclerosingpanencephalitis virus, distemper virus, Rinderpest virus), the genusPneumovirus (respiratory syncytial virus (RSV), Bovine respiratorysyncytial virus and Pneumonia virus of mice); forest virus, Sindbisvirus, Chikungunya virus, O'Nyong-Nyong virus. Ross river virus,Venezuelan equine encephalitis virus, Western equine encephalitisvirus), the genus Flavirius (Mosquito borne yellow fever virus, Denguevirus, Japanese encephalitis virus, St. Louis encephalitis virus. MurrayValley encephalitis virus, West Nile virus, Kunjin virus, CentralEuropean tick borne virus, Far Eastern tick borne virus, Kyasanur forestvirus, Louping III virus, Powassan virus, Omsk hemorrhagic fever virus),the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosaldisease virus, Hog cholera virus, Border disease virus); the familyBunyaviridae, including the genus Bunyvirus (Bunyamwera and relatedviruses, California encephalitis group viruses), the genus Phlebovirus(Sandfly fever Sicilian virus, Rift Valley fever virus), the genusNairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep diseasevirus), and the genus Uukuvirus (Uukuniemi and related viruses); thefamily Orthomyxoviridae, including the genus Influenza virus (Influenzavirus type A, many human subtypes); Swine influenza virus, and Avian andEquine Influenza viruses; influenza type B (many human subtypes), andinfluenza type C (possible separate genus); the family paramyxoviridae,including the genus Paramyxovirus (Parainfluenza virus type 1, Sendaivirus, Hemadsorption virus, Parainfluenza viruses types 2 to 5,Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measlesvirus, subacute sclerosing panencephalitis virus, distemper virus,Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus(RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice);the family Rhabdoviridae, including the genus Vesiculovirus (VSV),Chandipura virus, Flanders-Hart Park virus), the genus Lyssavirus(Rabies virus), fish Rhabdoviruses and, filoviruses (Marburg virus andEbola virus); the family Arenaviridae, including Lymphocyticchoriomeningitis virus (LCM), Tacaribe virus complex, and Lassa virus;the family Coronoaviridae, including Infectious Bronchitis Virus (IBV),Mouse Hepatitis virus, Human enteric corona virus, and Feline infectiousperitonitis (Feline coronavirus).

Illustrative DNA viruses that may be used as core particle include, butare not limited to: the family Poxyiridae, including the genusOrthopoxvirus (Variola major, Variola minor, Monkey pox Vaccinia,Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus(Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avianpoxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genusSuipoxvirus (Swinepox), the genus Parapoxvirus (contagious postulardermatitis virus, pseudocowpox, bovine papular stomatitis virus); thefamily Iridoviridae (African swine fever virus, Frog viruses 2 and 3,Lymphocystis virus of fish); the family Herpesviridae, including thealpha-Herpesviruses (Herpes Simplex Types 1 and 2, Varicella-Zoster,Equine abortion virus, Equine herpes virus 2 and 3, pseudorabies virus,infectious bovine keratoconjunctivitis virus, infectious bovinerhinotracheitis virus, feline rhinotracheitis virus, infectiouslaryngotracheitis virus) the Beta-herpesviruses (Human cytomegalovirusand cytomegaloviruses of swine, monkeys and rodents); thegamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease virus,Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus, guinea pigherpes virus, Lucke tumor virus); the family Adenoviridae, including thegenus Mastadenovirus (Human subgroups A, B, C, D and B and ungrouped;simian adenoviruses (at least 23 serotypes), infectious caninehepatitis, and adenoviruses of cattle, pigs, sheep, frogs and many otherspecies, the genus Aviadenovirus (Avian adenoviruses); andnon-cultivatable adenoviruses; the family Papoviridae, including thegenus Papillomavirus (Human papilloma viruses, bovine papilloma viruses,Shope rabbit papilloma virus, and various pathogenic papilloma virusesof other species), the genus Polyomavirus (polyomavirus, Simianvacuolating agent (SV-40), Rabbit vacuolating agent (RKV), K virus, BKvirus, JC virus, and other primate polyoma viruses such as Lymphotrophicpapilloma virus); the family Parvoviridae including the genusAdeno-associated viruses, the genus Parvovirus (Feline panleukopeniavirus, bovine parvovirus, canine parvovirus, Aleutian mink diseasevirus, etc.). Finally, DNA viruses may include viruses such as chronicinfectious neuropathic agents (CHINA virus).

In other embodiments, a bacterial pilin, a subportion of a bacterialpilin, or a fusion protein which contains either a bacterial pilin orsubportion thereof is used to prepare compositions and vaccinecompositions, respectively, of the invention. Examples of pilin proteinsinclude pilins produced by Escherichia coli, Haemophilus influenzae,Neisseria meningitidis, Neiseria gonorrhoeae, Caulobacter crescentus,Pseudomonas sturzeri, and Pseudomonas aeruginosa.

The amino acid sequences of pilin proteins suitable for use with thepresent invention include those set out in GenBank reports AJ000636,AJ132364, AF229646, AF051814, AF051815, and X00981, the entiredisclosures of which are incorporated herein by reference.

Bacterial pilin proteins are generally processed to remove N-terminalleader sequences prior to export of the proteins into the bacterialperiplasm. Further, as one skilled in the art would recognize, bacterialpilin proteins used to prepare compositions and vaccine compositions,respectively, of the invention will generally not have the naturallypresent leader sequence.

One specific example of a pilin protein suitable for use in the presentinvention is the P-pilin of E. coli (GenBank report AF237482 (SEQ IDNO:1)). An example of a Type-1 E. coli pilin suitable for use with theinvention is a pilin having the amino acid sequence set out in GenBankreport P04128 (SEQ ID NO:2), which is encoded by nucleic acid having thenucleotide sequence set out in GenBank report M27603 (SEQ ID NO:3). Theentire disclosures of these GenBank reports are incorporated herein byreference. Again, the mature form of the above referenced protein wouldgenerally be used to prepare compositions and vaccine compositions,respectively, of the invention.

Bacterial pilins or pilin subportions suitable for use in the practiceof the present invention will generally be able to associate to formordered and repetitive antigen arrays.

Methods for preparing pili and pilus-like structures in vitro are knownin the art, Bullitt et al., Proc. Natl. Acad. Sci. USA 93:12890-12895(1996), for example, describe the in vitro reconstitution of E. coliP-pili subunits. Furthermore, Eshdat et al., J. Bacteriol. 148:308-314(1981) describe methods suitable for dissociating Type-1 pill of E. coliand the reconstitution of pill. In brief, these methods are as follows:pili are dissociated by incubation at 37° C. in saturated guanidinehydrochloride. Pilin proteins are then purified by chromatography, afterwhich pilin dimers are formed by dialysis against 5 mMtris(hydroxymethyl)aminomethane hydrochloride (pH 8.0). Eshdat et al.also found that pilin dimers reassemble to form pili upon dialysisagainst the 5 mM tris(hydroxymethyl)aminomethane (pH 8.0) containing 5mM MgCl₂.

Further, using, for example, conventional genetic engineering andprotein modification methods, pilin proteins may be modified to containa first attachment site to which an antigen or antigenic determinant islinked through a second attachment site. Alternatively, antigens orantigenic determinants can be directly linked through a secondattachment site to amino acid residues which are naturally resident inthese proteins. These modified pilin proteins may then be used invaccine compositions of the invention.

Bacterial pilin proteins used to prepare compositions and vaccinecompositions, respectively, of the invention may be modified in a mannersimilar to that described herein for HBcAg. For example, cysteine andlysine residues may be either deleted or substituted with other aminoacid residues and first attachment sites may be added to these proteins.Further, pilin proteins may either be expressed in modified form or maybe chemically modified after expression. Similarly, intact pili may beharvested from bacteria and then modified chemically.

In another embodiment, pill or pilus-like structures are harvested frombacteria (e.g., E. coli) and used to form compositions and vaccinecompositions of the invention. One example of pill suitable forpreparing compositions and vaccine compositions is the Type-1 pilus ofE. coli, which is formed from pilin monomers having the amino acidsequence set out in SEQ ID NO:2.

A number of methods for harvesting bacterial pili are known in the art.Bullitt and Makowski (Biophys. J. 74:623-632 (1998)), for example,describe a pilus purification method for harvesting P-pili from E. coli.According to this method, pili are sheared from hyperpiliated E. colicontaining a P-pilus plasmid and purified by cycles of solubilizationand MgCl₂ (1.0 M) precipitation.

Once harvested, pili or pilus-like structures may be modified in avariety of ways. For example, a first attachment site can be added tothe pili to which antigens or antigen determinants may be attachedthrough a second attachment site. In other words, bacterial pili orpilus-like structures can be harvested and modified to lead to orderedand repetitive antigen arrays.

Antigens or antigenic determinants could be linked to naturallyoccurring cysteine resides or lysine residues present in Pili orpilus-like structures. In such instances, the high order andrepetitiveness of a naturally occurring amino acid residue would guidethe coupling of the antigens or antigenic determinants to the pill orpilus-like structures. For example, the pill or pilus-like structurescould be linked to the second attachment sites of the antigens orantigenic determinants using a heterobifunctional cross-linking agent.

When structures which are naturally synthesized by organisms (e.g.,pill) are used to prepare compositions and vaccine compositions of theinvention, it will often be advantageous to genetically engineer theseorganisms so that they produce structures having desirablecharacteristics. For example, when Type-1 pili of E. coli are used, theE. coli from which these pili are harvested may be modified so as toproduce structures with specific characteristics. Examples of possiblemodifications of pilin proteins include the insertion of one or morelysine residues, the deletion or substitution of one or more of thenaturally resident lysine residues, and the deletion or substitution ofone or more naturally resident cysteine residues (e.g., the cysteineresidues at positions 44 and 84 in SEQ ID NO:2).

Further, additional modifications can be made to pilin genes whichresult in the expression products containing a first attachment siteother than a lysine residue (e.g., a FOS or JUN domain). Of course,suitable first attachment sites will generally be limited to those whichdo not prevent pilin proteins from forming pill or pilus-like structuressuitable for use in vaccine compositions of the invention.

Pilin genes which naturally reside in bacterial cells can be modified invivo (e.g., by homologous recombination) or pilin genes with particularcharacteristics can be inserted into these cells. For examples, pilingenes could be introduced into bacterial cells as a component of eithera replicable cloning vector or a vector which inserts into the bacterialchromosome. The inserted pilin genes may also be linked to expressionregulatory control sequences (e.g., a lac operator).

In most instances, the pili or pilus-like structures used incompositions and vaccine compositions, respectively, of the inventionwill be composed of single type of a pilin subunit. Pili or pilus-likestructures composed of identical subunits will generally be used becausethey are expected to firm structures which present highly ordered andrepetitive antigen arrays.

However, the compositions of the invention also include compositions andvaccines comprising pill or pilus-like structures formed fromheterogenous pilin subunits. The pilin subunits which form these pill orpilus-like structures can be expressed from genes naturally resident inthe bacterial cell or may be introduced into the cells. When a naturallyresident pilin gene and an introduced gene are both expressed in a cellwhich forms pili or pilus-like structures, the result will generally bestructures formed from a mixture of these pilin proteins. Further, whentwo or more pilin genes are expressed in a bacterial cell, the relativeexpression of each pilin gene will typically be the factor whichdetermines the ratio of the different pilin subunits in the pill orpilus-like structures.

When pill or pilus-like structures having a particular composition ofmixed pilin subunits is desired, the expression of at least one of thepilin genes can be regulated by a heterologous, inducible promoter. Suchpromoters, as well as other genetic elements, can be used to regulatethe relative amounts of different pilin subunits produced in thebacterial cell and, hence, the composition of the pili or pilus-likestructures.

In additional, the antigen or antigenic determinant can be linked tobacterial pill or pilus-like structures by a bond which is not a peptidebond, bacterial cells which produce pill or pilus-like structures usedin the compositions of the invention can be genetically engineered togenerate pilin proteins which are fused to an antigen or antigenicdeterminant. Such fusion proteins which form pili or pilus-likestructures are suitable for use in vaccine compositions of theinvention.

In a preferred embodiment, the core particle is a virus-like particle,wherein the virus-like particle is a recombinant virus-like particle.The skilled artisan can produce VLPs using recombinant DNA technologyand virus coding sequences which are readily available to the public.For example, the coding sequence of a virus envelope or core protein canbe engineered for expression in a baculovirus expression vector using acommercially available baculovirus vector, under the regulatory controlof a virus promoter, with appropriate modifications of the sequence toallow functional linkage of the coding sequence to the regulatorysequence. The coding sequence of a virus envelope or core protein canalso be engineered for expression in a bacterial expression vector, forexample.

Examples of VLPs include, but are not limited to, the capsid proteins ofHepatitis B virus (Ulrich, et al., Virus Res. 50:141-182 (1998)),measles virus (Warnes, et al., Gene 160; 173-178 (1995)), Sindbis virus,rotavirus (U.S. Pat. No. 5,071,651 and U.S. Pat. No. 5,374,426),foot-and-mouth-disease virus (Twomey, et al., Vaccine 13:1603-1610,(1995)), Norwalk virus (Jiang, X., et al., Science 250:1580-1583 (1990);Matsui, S. M., et al., J. Clin. Invest. 87:1456-1461 (1991)), theretroviral GAG protein (WO 96/30523), the retrotransposon Ty protein p1,the surface protein of Hepatitis B virus (WO 92/11291), human papillomavirus (WO 98/15631), RNA phages, Ty, fr-phage, GA-phage, AP205-phage andQβ-phage.

As will be readily apparent to those skilled in the art, the VLP of theinvention is not limited to any specific form. The particle can besynthesized chemically or through a biological process, which can benatural or non-natural. By way of example, this type of embodimentincludes a virus-like particle or a recombinant form thereof.

In a more specific embodiment, the VLP can comprise, or alternativelyessentially consist of, or alternatively consist of recombinantpolypeptides, or fragments thereof, being selected from recombinantpolypeptides of Rotavirus, recombinant polypeptides of Norwalk virus,recombinant polypeptides of Alphavirus, recombinant polypeptides of Footand Mouth Disease virus, recombinant polypeptides of measles virus,recombinant polypeptides of Sindbis virus, recombinant polypeptides ofPolyoma virus, recombinant polypeptides of Retrovirus, recombinantpolypeptides of Hepatitis B virus (e.g., a HBcAg), recombinantpolypeptides of Tobacco mosaic virus, recombinant polypeptides of FlockHouse Virus, recombinant polypeptides of human Papillomavirus,recombinant polypeptides of bacteriophages, recombinant polypeptides ofRNA phages, recombinant polypeptides of Ty, recombinant polypeptides offr-phage, recombinant polypeptides of GA-phage and recombinantpolypeptides of Qβ-phage. The virus-like particle can further comprise,or alternatively essentially consist of, or alternatively consist of,one or more fragments of such polypeptides, as well as variants of suchpolypeptides. Variants of polypeptides can share, for example, at least80%, 85%, 90%, 95%, 97%, or 99% identity at the amino acid level withtheir wild-type counterparts.

In a preferred embodiment, the virus-like particle comprises, consistsessentially of, or alternatively consists of recombinant proteins, orfragments thereof, of a RNA-phage. Preferably, the RNA-phage is selectedfrom the group consisting of a) bacteriophage Qβ; b) bacteriophage R17;c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f)bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i)bacteriophage NL95; k) bacteriophage f2; l) bacteriophage PP7, and m)bacteriophage AP205.

In another preferred embodiment of the present invention, the virus-likeparticle comprises, or alternatively consists essentially of, oralternatively consists of recombinant proteins, or fragments thereof, ofthe RNA-bacteriophage Qβ or of the RNA-bacteriophage fr, or of theRNA-bacteriophage AP205.

In a further preferred embodiment of the present invention, therecombinant proteins comprise, or alternatively consist essentially of,or alternatively consist of coat proteins of RNA phages.

RNA-phage coat proteins forming capsids or VLPs, or fragments of thebacteriophage coat proteins compatible with self-assembly into a capsidor a VLP, are, therefore, further preferred embodiments of the presentinvention. Bacteriophage Qβ coat proteins, for example, can be expressedrecombinantly in E. coli. Further, upon such expression these proteinsspontaneously form capsids. Additionally, these capsids form a structurewith an inherent repetitive organization.

Specific preferred examples of bacteriophage coat proteins which can beused to prepare compositions of the invention include the coat proteinsof RNA bacteriophages such as bacteriophage Qβ (SEQ ID NO:4; PIRDatabase, Accession No. VCBPQβ referring to Qβ CP and SEQ ID NO: 5;Accession No. AAA16663 referring to Qβ A1 protein), bacteriophage R17(SEQ ID NO:6; PIR Accession No. VCBPR7), bacteriophage fr (SEQ ID NO:7;PIR Accession No. VCBPFR), bacteriophage GA (SEQ ID NO:8; GenBankAccession No. NP-040754), bacteriophage SP (SEQ ID NO:9; GenBankAccession No. CAA30374 referring to SP CP and SEQ ID NO: 10; AccessionNo. NP 695026 referring to SP A1 protein), bacteriophage MS2 (SEQ ID NO:11; PIR Accession No. VCBPM2), bacteriophage M11 (SEQ ID NO:12; GenBankAccession No. AAC06250), bacteriophage MX1 (SEQ ID NO:13; GenBankAccession No. AAC14699), bacteriophage NL95 (SEQ ID NO:14; GenBankAccession No. AAC14704), bacteriophage f2 (SEQ ID NO: 15; GenBankAccession No. P03611), bacteriophage PP7 (SEQ ID NO: 16), andbacteriophage AP205 (SEQ ID NO: 28). Furthermore, the A1 protein ofbacteriophage Qβ (SEQ ID NO: 5) or C-terminal truncated forms missing asmuch as 100, 150 or 180 amino acids from its C-terminus may beincorporated in a capsid assembly of Qβ coat proteins. Generally, thepercentage of Qβ A1 protein relative to Qβ CP in the capsid assemblywill be limited, in order to ensure capsid formation.

Qβ coat protein has also been found to self-assemble into capsids whenexpressed in E. coli (Kozlovska T M. et al., GENE 137: 133-137 (1993)).The obtained capsids or virus-like particles showed an icosahedralphage-like capsid structure with a diameter of 25 nm and T=3 quasisymmetry. Further, the crystal structure of phage Qβ has been solved.The capsid contains 180 copies of the coat protein, which are linked incovalent pentamers and hexamers by disulfide bridges (Golmohammadi, R.et al. Structure 4: 543-5554 (1996)) leading to a remarkable stabilityof the capsid of Qβ coat protein. Capsids or VLPs made from recombinantQβ coat protein may contain, however, subunits not linked via disulfidelinks to other subunits within the capsid, or incompletely linked.However, typically more than about 80% of the subunits are linked viadisulfide bridges to each other within the VLP. Thus, upon loadingrecombinant Qβ capsid on non-reducing SDS-PAGE, bands corresponding tomonomeric Qβ coat protein as well as bands corresponding to the hexameror pentamer of Qβ coat protein are visible. Incompletelydisulfide-linked subunits could appear as dimer, trimer or even tetramerbands in non-reducing SDS-PAGE. Qβ capsid protein also shows unusualresistance to organic solvents and denaturing agents. Surprisingly, wehave observed that DMSO and acetonitrile concentrations as high as 30%,and Guanidinium concentrations as high as 1 M do not affect thestability of the capsid. The high stability of the capsid of Qβ coatprotein is an advantageous feature, in particular, for its use inimmunization and vaccination of mammals and humans in accordance of thepresent invention.

Upon expression in E. coli, the N-terminal methionine of Qβ coat proteinis usually removed, as we observed by N-terminal Edman sequencing asdescribed in Stoll, E. et al. J. Biol. Chem. 252:990-993 (1977). VLPcomposed from Qβ coat proteins where the N-terminal methionine has notbeen removed, or VLPs comprising a mixture of Qβ coat proteins where theN-terminal methionine is either cleaved or present are also within thescope of the present invention.

Further preferred virus-like particles of RNA-phages, in particular ofQβ, in accordance of this invention are disclosed in WO 02/056905, thedisclosure of which is herewith incorporated by reference in itsentirety.

Further RNA phage coat proteins have also been shown to self-assembleupon expression in a bacterial host (Kastelein, R A. et al., Gene 23:245-254 (1983), Kozlovskaya, T M. et al., Dokl. Akad. Nauk SSSR 287:452-455 (1986), Adhin, M R. et al., Virology 170: 238-242 (1989), Ni, CZ., et al., Protein Sci. 5: 2485-2493 (1996), Priano, C. et al., J. Mol.Biol. 249: 283-297 (1995)). The Qβ phage capsid contains, in addition tothe coat protein, the so called read-through protein A1 and thematuration protein A2. A1 is generated by suppression at the UGA stopcodon and has a length of 329 aa. The capsid of phage Qβ recombinantcoat protein used in the invention is devoid of the A2 lysis protein,and contains RNA from the host. The coat protein of RNA phages is an RNAbinding protein, and interacts with the stem loop of the ribosomalbinding site of the replicase gene acting as a translational repressorduring the life cycle of the virus. The sequence and structural elementsof the interaction are known (Witherell, G W. & Uhlenbeck, O C.Biochemistry 28: 71-76 (1989); Lim F. et al., J. Biol. Chem. 271:31839-31845 (1996)). The stem loop and RNA in general are known to beinvolved in the virus assembly (Golmohammadi, R. et al., Structure 4:543-5554 (1996)).

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively consists essentially of,or alternatively consists of recombinant proteins, or fragments thereof,of a RNA-phage, wherein the recombinant proteins comprise, alternativelyconsist essentially of or alternatively consist of mutant coat proteinsof a RNA phage, preferably of mutant coat proteins of the RNA phagesmentioned above. In another preferred embodiment, the mutant coatproteins of the RNA phage have been modified by removal of at least one,or alternatively at least two, lysine residue by way of substitution, orby addition of at least one lysine residue by way of substitution:alternatively, the mutant coat proteins of the RNA phage have beenmodified by deletion of at least one, or alternatively at least two,lysine residue, or by addition of at least one lysine residue by way ofinsertion. The deletion, substitution or addition of at least one lysineresidue allows varying the degree of coupling, i.e. the amount of Aβ1-6peptides per subunits of the VLP of the RNA-phages, in particular, tomatch and tailor the requirements of the vaccine.

In another preferred embodiment, the virus-like particle comprises, oralternatively consists essentially of, or alternatively consists ofrecombinant proteins, or fragments thereof, of the RNA-bacteriophage Qβwherein the recombinant proteins comprise, or alternatively consistessentially of, or alternatively consist of coat proteins having anamino acid sequence of SEQ ID NO:4, or a mixture of coat proteins havingamino acid sequences of SEQ ID NO:4 and of SEQ ID NO: 5 or mutants ofSEQ ID NO: 5 and wherein the N-terminal methionine is preferablycleaved.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, consists essentially of or alternativelyconsists of recombinant proteins of Qβ, or fragments thereof, whereinthe recombinant proteins comprise, or alternatively consist essentiallyof, or alternatively consist of mutant Qβ coat proteins. In anotherpreferred embodiment, these mutant coat proteins have been modified byremoval of at least one lysine residue by way of substitution, or byaddition of at least one lysine residue by way of substitution.Alternatively, these mutant coat proteins have been modified by deletionof at least one lysine residue, or by addition of at least one lysineresidue by way of insertion.

Four lysine residues are exposed on the surface of the capsid of Qβ coatprotein. Q mutants, for which exposed lysine residues are replaced byarginines can also be used for the present invention. The following Qβcoat protein mutants and mutant Qβ VLPs can, thus, be used in thepractice of the invention: “Qβ-240” (Lys13-Arg; SEQ ID NO:17), “Qβ-243”(Asn 10-Lys; SEQ ID NO:18), “Qβ-250” (Lys 2-Arg, Lys13-Arg; SEQ IDNO:19), “Qβ-251” (SEQ ID NO:20) and “Qβ-259” (Lys 2-Arg, Lys16-Arg; SEQID NO:21). Thus, in further preferred embodiment of the presentinvention, the virus-like particle comprises, consists essentially of oralternatively consists of recombinant proteins of mutant Qβ coatproteins, which comprise proteins having an amino acid sequence selectedfrom the group of a) the amino acid sequence of SEQ ID NO: 17; b) theamino acid sequence of SEQ ID NO: 18; c) the amino acid sequence of SEQID NO: 19; d) the amino acid sequence of SEQ ID NO:20; and e) the aminoacid sequence of SEQ ID NO: 21. The construction, expression andpurification of the above indicated Qβ coat proteins, mutant Qβ coatprotein VLPs and capsids, respectively, are described in WO 02/056905.In particular is hereby referred to Example 18 of above mentionedapplication.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively consists essentially of,or alternatively consists of recombinant proteins of Qβ, or fragmentsthereof, wherein the recombinant proteins comprise, consist essentiallyof or alternatively consist of a mixture of either one of the foregoingQβ mutants and the corresponding A1 protein.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant proteins, or fragments thereof,of RNA-phage AP205.

The AP205 genome consists of a maturation protein, a coat protein, areplicase and two open reading frames not present in related phages; alysis gene and an open reading frame playing a role in the translationof the maturation gene (Klovins J., et al., J. Gen. Viral. 83: 1523-33(2002)). AP205 coat protein can be expressed from plasmid pAP283-58 (SEQID NO: 27), which is a derivative of pQb10 (Kozlovska, T. M. et al.,Gene 137:133-37 (1993)), and which contains an AP205 ribosomal bindingsite. Alternatively, AP205 coat protein may be cloned into pQb185,downstream of the ribosomal binding site present in the vector. Bothapproaches lead to expression of the protein and formation of capsids asdescribed in the co-pending US provisional patent application with thetitle “Molecular Antigen Arrays” (Atty. Docket No. 1700.0310000) andhaving been filed on Jul. 17, 2002, which is incorporated by referencein its entirety. Vectors pQh10 and pQb185 are vectors derived from pGEMvector, and expression of the cloned genes in these vectors iscontrolled by the trp promoter (Kozlovska, T. M. et al., Gene 137:133-37(1993)). Plasmid pAP283-58 (SEQ ID NO:27) comprises a putative AP205ribosomal binding site in the following sequence, which is downstream ofthe XbaI site, and immediately upstream of the ATG start codon of theAP205 coat protein: tctagaATTTTCTGCGCACCCATCCCGGGTGGCGCCCAAAGTGAGGAAAATCACatg (SEQ ID NO:57). The vector pQb185comprises a Shine Delagarno sequence downstream from the XbaI site andupstream of the start codon (rctagaTTAACCCAACGCGTAGGAG TCAGGCCaig (SEQID NO:58), Shine Delagarno sequence underlined).

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant coat proteins, or fragmentsthereof, of the RNA-phage AP205.

This preferred embodiment of the present invention, thus, comprisesAP205 coat proteins that form capsids. Such proteins are recombinantlyexpressed, or prepared from natural sources. AP205 coat proteinsproduced in bacteria spontaneously form capsids, as evidenced byElectron Microscopy (EM) and immunodiffusion. The structural propertiesof the capsid formed by the AP205 coat protein (SEQ ID NO: 28) and thoseformed by the coat protein of the AP205 RNA phage are nearlyindistinguishable when seen in EM. AP205 VLPs are highly immunogenic,and can be linked with antigens and/or antigenic determinants togenerate vaccine constructs displaying the antigens and/or antigenicdeterminants oriented in a repetitive manner. High titers are elicitedagainst the so displayed antigens showing that bound antigens and/orantigenic determinants are accessible for interacting with antibodymolecules and are immunogenic.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant mutant coat proteins, orfragments thereof, of the RNA-phage AP205.

Assembly-competent mutant forms of AP205 VLPs, including AP205 coatprotein with the substitution of proline at amino acid 5 to threonine(SEQ ID NO: 29), may also be used in the practice of the invention andleads to a further preferred embodiment of the invention. These VLPs,AP205 VLPs derived from natural sources, or AP205 viral particles, maybe bound to antigens to produce ordered repetitive arrays of theantigens in accordance with the present invention.

AP205 PS-T mutant coat protein can be expressed from plasmid pAP281-32(SEQ ID No. 30), which is derived directly from pQb185, and whichcontains the mutant AP205 coat protein gene instead of the Qβ coatprotein gene. Vectors for expression of the AP205 coat protein aretransfected into E. coli for expression of the AP205 coat protein.

Methods for expression of the coat protein and the mutant coat protein,respectively, leading to the self-assembly into VLPs are described inExample 1. Suitable E. coli strains include, but are not limited to, E.coli K802, JM 109, RR1. Suitable vectors and strains and combinationsthereof can be identified by testing expression of the coat protein andmutant coat protein, respectively, by SDS-PAGE and capsid formation andassembly by optionally first purifying the capsids by gel filtration andsubsequently testing them in an immunodiffusion assay (Ouchterlony test)or Electron Microscopy (Kozlovska, T. M. et al., Gene 137:133-37(1993)).

AP205 coat proteins expressed from the vectors pAP283-58 and pAP281-32may be devoid of the initial Methionine amino-acid, due to processing inthe cytoplasm of E. coli. Cleaved, uncleaved forms of AP205 VLP ormixtures thereof are further preferred embodiments of the invention.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of a mixture of recombinant coat proteins, orfragments thereof, of the RNA-phage AP205 and of recombinant mutant coatproteins, or fragments thereof, of the RNA-phage AP205.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of fragments of recombinant coat proteins orrecombinant mutant coat proteins of the RNA-phage AP205.

Recombinant AP205 coat protein fragments capable of assembling into aVLP and a capsid, respectively are also useful in the practice of theinvention. These fragments may be generated by deletion, eitherinternally or at the termini of the coat protein and mutant coatprotein, respectively. Insertions in the coat protein and mutant coatprotein sequence or fusions of antigen sequences to the coat protein andmutant coat protein sequence, and compatible with assembly into a VLP,are further embodiments of the invention and lead to chimeric AP205 coatproteins, and particles, respectively. The outcome of insertions,deletions and fusions to the coat protein sequence and whether it iscompatible with assembly into a VLP can be determined by electronmicroscopy.

The particles formed by the AP205 coat protein, coat protein fragmentsand chimeric coat proteins described above, can be isolated in pure formby a combination of fractionation steps by precipitation and ofpurification steps by gel filtration using e.g. Sepharose CL-4B,Sepharose CL-2B, Sepharose CL-6B columns and combinations thereof. Othermethods of isolating virus-like particles are known in the art, and maybe used to isolate the virus-like particles (VLPs) of bacteriophageAP205. For example, the use of ultracentrifugation to isolate VLPs ofthe yeast retrotransposon Ty is described in U.S. Pat. No. 4,918,166,which is incorporated by reference herein in its entirety.

The crystal structure of several RNA bacteriophages has been determined(Golmohammadi, R. et al., Structur 4:543-554 (1996)). Using suchinformation, surface exposed residues can be identified and, thus,RNA-phage coat proteins can be modified such that one or more reactiveamino acid residues can be inserted by way of insertion or substitution.As a consequence, those modified forms of bacteriophage coat proteinscan also be used for the present invention. Thus, variants of proteinswhich form capsids or capsid-like structures (e.g., coat proteins ofbacteriophage Qβ, bacteriophage R17, bacteriophage fr, bacteriophage GA,bacteriophage SP, bacteriophage AP205, and bacteriophage MS2) can alsobe used to prepare compositions of the present invention.

Although the sequence of the variants proteins discussed above willdiffer from their wild-type counterparts, these variant proteins willgenerally retain the ability to form capsids or capsid-like structures.Thus, the invention further includes compositions and vaccinecompositions, respectively, which further includes variants of proteinswhich form capsids or capsid-like structures, as well as methods forpreparing such compositions and vaccine compositions, respectively,individual protein subunits used to prepare such compositions, andnucleic acid molecules which encode these protein subunits. Thus,included within the scope of the invention are variant forms ofwild-type proteins which form capsids or capsid-like structures andretain the ability to associate and form capsids or capsid-likestructures.

As a result, the invention further includes compositions and vaccinecompositions, respectively, comprising proteins, which comprise, oralternatively consist essentially of, or alternatively consist of aminoacid sequences which are at least 80%, 85%, 90%, 95%, 97%, or 99%identical to wild-type proteins which form ordered arrays and have aninherent repetitive structure, respectively.

Further included within the scope of the invention are nucleic acidmolecules which encode proteins used to prepare compositions of thepresent invention.

In other embodiments, the invention further includes compositionscomprising proteins, which comprise, or alternatively consistessentially of, or alternatively consist of amino acid sequences whichare at least 80%, 85%, 90%, 95%, 97%, or 99% identical to any of theamino acid sequences shown in SEQ ID NOs:4-21.

Proteins suitable for use in the present invention also includeC-terminal truncation mutants of proteins which form capsids orcapsid-like structures, or VLPs. Specific examples of such truncationmutants include proteins having an amino acid sequence shown in any ofSEQ ID NOs:4-21 where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acidshave been removed from the C-terminus. Typically, theses C-terminaltruncation mutants will retain the ability to form capsids orcapsid-like structures.

Further proteins suitable for use in the present invention also includeN-terminal truncation mutants of proteins which form capsids orcapsid-like structures. Specific examples of such truncation mutantsinclude proteins having an amino acid sequence shown in any of SEQ IDNOs:4-21 where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids havebeen removed from the N-terminus. Typically, these N-terminal truncationmutants will retain the ability to form capsids or capsid-likestructures.

Additional proteins suitable for use in the present invention include N-and C-terminal truncation mutants which form capsids or capsid-likestructures. Suitable truncation mutants include proteins having an aminoacid sequence shown in any of SEQ ID NOs:4-21 where 1, 2, 5, 7, 9, 10,12, 14, 15, or 17 amino acids have been removed from the N-terminus and1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids have been removed fromthe C-terminus. Typically, these N-terminal and C-terminal truncationmutants will retain the ability to form capsids or capsid-likestructures.

The invention further includes compositions comprising proteins whichcomprise, or alternatively consist essentially of, or alternativelyconsist of, amino acid sequences which are at least 80%, 85%, 90%, 95%,97%, or 99% identical to the above described truncation mutants.

The invention thus includes compositions and vaccine compositionsprepared from proteins which form capsids or VLPs, methods for preparingthese compositions from individual protein subunits and VLPs or capsids,methods for preparing these individual protein subunits, nucleic acidmolecules which encode these subunits, and methods for vaccinatingand/or eliciting immunological responses in individuals using thesecompositions of the present invention.

As previously stated, the invention includes virus-like particles orrecombinant forms thereof. In one further preferred embodiment, theparticles used in compositions of the invention are composed of aHepatitis B core protein (—HBcAg) or a fragment of a HBcAg. In a furtherembodiment, the particles used in compositions of the invention arecomposed of a Hepatitis B core protein (HBcAg) or a fragment of a HBcAgprotein, which has been modified to either eliminate or reduce thenumber of free cysteine residues. Zhou et al. (J. Virol. 66:5393-5398(1992)) demonstrated that HBcAgs which have been modified to remove thenaturally resident cysteine residues retain the ability to associate andform capsids. Thus, VLPs suitable for use in compositions of theinvention include those comprising modified HBcAgs, or fragmentsthereof, in which one or more of the naturally resident cysteineresidues have been either deleted or substituted with another amino acidresidue (e.g., a serine residue).

The HBcAg is a protein generated by the processing of a Hepatitis B coreantigen precursor protein. A number of isotypes of the HBcAg have beenidentified and their amino acids sequences are readily available tothose skilled in the art. In most instances, compositions and vaccinecompositions, respectively, of the invention will be prepared using theprocessed form of a HBcAg (i.e., a HBcAg from which the N-terminalleader sequence of the Hepatitis B core antigen precursor protein havebeen removed).

Further, when HBcAgs are produced under conditions where processing willnot occur, the HBcAgs will generally be expressed in “processed” form.For example, when an E. coli expression system directing expression ofthe protein to the cytoplasm is used to produce HBcAgs of the invention,these proteins will generally be expressed such that the N-terminalleader sequence of the Hepatitis B core antigen precursor protein is notpresent.

The preparation of Hepatitis B virus-like particles, which can be usedfor the present invention, is disclosed, for example, in WO 00/32227,and hereby in particular in Examples 17 to 19 and 21 to 24, as well asin WO 01/85208, and hereby in particular in Examples 17 to 19, 21 to 24,31 and 41, and in WO 02/056905. For the latter application, it is inparticular referred to Example 23, 24, 31 and 51. All three documentsare explicitly incorporated herein by reference.

The present invention also includes HBcAg variants which have beenmodified to delete or substitute one or more additional cysteineresidues. It is known in the art that free cysteine residues can beinvolved in a number of chemical side reactions. These side reactionsinclude disulfide exchanges, reaction with chemical substances ormetabolites that are, for example, injected or formed in a combinationtherapy with other substances, or direct oxidation and reaction withnucleotides upon exposure to UV light. Toxic adducts could thus begenerated, especially considering the fact that HBcAgs have a strongtendency to bind nucleic acids. The toxic adducts would thus bedistributed between a multiplicity of species, which individually mayeach be present at low concentration, but reach toxic levels whentogether.

In view of the above, one advantage to the use of HBcAgs in vaccinecompositions which have been modified to remove naturally residentcysteine residues is that sites to which toxic species can bind whenantigens or antigenic determinants are attached would be reduced innumber or eliminated altogether.

A number of naturally occurring HBcAg variants suitable for use in thepractice of the present invention have been identified. Yuan et al. (J.Virol. 73; 10122-10128 (1999)), for example, describe variants in whichthe isoleucine residue at position corresponding to position 97 in SEQID NO:22 is replaced with either a leucine residue or a phenylalanineresidue. The amino acid sequences of a number of HBcAg variants, as wellas several Hepatitis B core antigen precursor variants, are disclosed inGenBank reports AAF121240, AF121239, X85297, X02496, X85305, X85303,AF151735, X85259, X85286, X85260, X85317, X85298, AF043593, M20706,X85295, X80925, X85284, X85275, X72702, X85291, X65258, X85302, M32138,X85293, X85315, U95551, X85256, X85316, X85296, AB033559, X59795,X85299, X85307, X65257, X85311, X85301 (SEQ ID NO:23), X85314, X85287,X85272, X85319, Aβ010289, X85285, Aβ010289, AF121242, M90520 (SEQ IDNO:24), P03153, AF110999, and M95589, the disclosures of each of whichare incorporated herein by reference. The sequences of the hereinabovementioned Hepatitis B core antigen precursor variants are furtherdisclosed in WO 01/85208 in SEQ ID NOs: 89-138. These HBcAg variantsdiffer in amino acid sequence at a number of positions, including aminoacid residues which corresponds to the amino acid residues located atpositions 12, 13, 21, 22, 24, 29, 32, 33, 35, 38, 40, 42, 44, 45, 49,51, 57, 58, 59, 64, 66, 67, 69, 74, 77, 80, 81, 87, 92, 93, 97, 98, 100,103, 105, 106, 109, 113, 116, 121, 126, 130, 133, 135, 141, 147, 149,157, 176, 178, 182 and 183 in SEQ ID NO:25. Further HBcAg variantssuitable for use in the compositions of the invention, and which may befurther modified according to the disclosure of this specification aredescribed in WO 00/198333, WO 00/177158 and WO 00/214478.

As noted above, generally processed HBcAgs (i.e., those which lackleader sequences) will be used in the compositions and vaccinecompositions, respectively, of the invention. The present inventionincludes vaccine compositions, as well as methods for using thesecompositions, which employ the above described variant HBcAgs.

Whether the amino acid sequence of a polypeptide has an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical toone of the above wild-type amino acid sequences, or a subportionthereof, can be determined conventionally using known computer programssuch the Bestfit program. When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference amino acid sequence, theparameters are set such that the percentage of identity is calculatedover the full length of the reference amino acid sequence and that gapsin homology of up to 5% of the total number of amino acid residues inthe reference sequence are allowed.

The amino acid sequences of the hereinabove mentioned HBcAg variants andprecursors are relatively similar to each other. Thus, reference to anamino acid residue of a HBcAg variant located at a position whichcorresponds to a particular position in SEQ ID NO:25, refers to theamino acid residue which is present at that position in the amino acidsequence shown in SEQ ID NO:25. The homology between these HBcAgvariants is for the most part high enough among Hepatitis B viruses thatinfect mammals so that one skilled in the art would have littledifficulty reviewing both the amino acid sequence shown in SEQ ID NO:25and that of a particular HBcAg variant and identifying “corresponding”amino acid residues. Furthermore, the HBcAg amino acid sequence shown inSEQ ID NO:24, which shows the amino acid sequence of a HBcAg derivedfrom a virus which infect woodchucks, has enough homology to the HBcAghaving the amino acid sequence shown in SEQ ID NO:25 that it is readilyapparent that a three amino acid residue insert is present in SEQ IDNO:25 between amino acid residues 155 and 156 of SEQ ID NO:25.

The invention also includes vaccine compositions which comprise HBcAgvariants of Hepatitis B viruses which infect birds, as wells as vaccinecompositions which comprise fragments of these HBcAg variants. For theseHBcAg variants one, two, three or more of the cysteine residuesnaturally present in these polypeptides could be either substituted withanother amino acid residue or deleted prior to their inclusion invaccine compositions of the invention.

As discussed above, the elimination of free cysteine residues reducesthe number of sites where toxic components can bind to the HBcAg, andalso eliminates sites where cross-linking of lysine and cysteineresidues of the same or of neighboring HBcAg molecules can occur.Therefore, in another embodiment of the present invention, one or morecysteine residues of the Hepatitis B virus capsid protein have beeneither deleted or substituted with another amino acid residue.

In other embodiments, compositions and vaccine compositions,respectively, of the invention will contain HBcAgs from which theC-terminal region (e.g., amino acid residues 145-185 or 150-185 of SEQID NO: 25) has been removed. Thus, additional modified HBcAgs suitablefor use in the practice of the present invention include C-terminaltruncation mutants. Suitable truncation mutants include HBcAgs where 1,5, 10, 15, 20, 25, 30, 34, 35, amino acids have been removed from theC-terminus.

HBcAgs suitable for use in the practice of the present invention alsoinclude N-terminal truncation mutants. Suitable truncation mutantsinclude modified HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 aminoacids have been removed from the N-terminus.

Further HBcAgs suitable for use in the practice of the present inventioninclude N- and C-terminal truncation mutants. Suitable truncationmutants include HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, and 17 aminoacids have been removed from the N-terminus and 1, 5, 10, 15, 20, 25,30, 34, 35 amino acids have been removed from the C-terminus.

The invention further includes compositions and vaccine compositions,respectively, comprising HBcAg polypeptides comprising, or alternativelyessentially consisting of, or alternatively consisting of, amino acidsequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identicalto the above described truncation mutants.

In certain embodiments of the invention, a lysine residue is introducedinto a HBcAg polypeptide, to mediate the binding of the Aβ1-6 peptide tothe VLP of HBcAg. In preferred embodiments, compositions of theinvention are prepared using a HBcAg comprising, or alternativelyconsisting of, amino acids 1-144, or 1-149, 1-185 of SEQ ID NO:25, whichis modified so that the amino acids corresponding to positions 79 and 80are replaced with a peptide having the amino acid sequence ofGly-Gly-Lys-Gly-Gly (SEQ ID NO:33) resulting in the HBcAg polypeptidehaving the sequence shown in SEQ ID NO: 26. These compositions areparticularly useful in those embodiments where an antigenic determinantis coupled to a VLP of HBcAg. In further preferred embodiments, thecysteine residues at positions 48 and 107 of SEQ ID NO:25 are mutated toserine. The invention further includes compositions comprising thecorresponding polypeptides having amino acid sequences shown in any ofthe hereinabove mentioned Hepatitis B core antigen precursor variantswhich also have above noted amino acid alterations. Further includedwithin the scope of the invention are additional HBcAg variants whichare capable of associating to form a capsid or VLP and have the abovenoted amino acid alterations. Thus, the invention further includescompositions and vaccine compositions, respectively, comprising HBcAgpolypeptides which comprise, or alternatively consist of, amino acidsequences which are at least 80%, 85%, 90%, 95%, 97% or 99% identical toany of the wild-type amino acid sequences, and forms of these proteinswhich have been processed, where appropriate, to remove the N-terminalleader sequence and modified with above noted alterations.

Compositions or vaccine compositions of the invention may comprisemixtures of different HBcAgs. Thus, these vaccine compositions may becomposed of HBcAgs which differ in amino acid sequence. For example,vaccine compositions could be prepared comprising a “wild-type” HBcAgand a modified HBcAg in which one or more amino acid residues have beenaltered (e.g., deleted, inserted or substituted). Further, preferredvaccine compositions of the invention are those which present highlyordered and repetitive antigen arrays, wherein the antigen is a Aβ1-6peptide.

In a further preferred embodiment of the present invention, the at leastone Aβ1-6 peptide is bound to said virus-like particle and coreparticle, respectively, by at least one covalent bond. Preferably, theleast one Aβ1-6 peptide is bound to the virus-like particle and coreparticle, respectively, by at least one covalent bond, said covalentbond being a non-peptide bond leading to a Aβ1-6 peptide array and Aβ1-6peptide-VLP conjugate, respectively. This Aβ1-6 peptide array andconjugate, respectively, has typically and preferably a repetitive andordered structure since the at least one Aβ1-6 peptide is bound to theVLP and core particle, respectively, in an oriented manner. Theformation of a repetitive and ordered Aβ1-6 peptide-VLP array andconjugate, respectively, is ensured by an oriented and directed as wellas defined binding and attachment, respectively, of the at least oneAβ1-6 peptide to the VLP and core particle, respectively, as will becomeapparent in the following. Furthermore, the typical inherent highlyrepetitive and organized structure of the VLPs and core particles,respectively, advantageously contributes to the display of the Aβ1-6peptide in a highly ordered and repetitive fashion leading to a highlyorganized and repetitive Aβ1-6 peptide-VLP array and conjugate,respectively.

Therefore, the preferred inventive conjugates and arrays, respectively,differ from prior art conjugates in their highly organized structure,dimensions, and in the repetitiveness of the antigen on the surface ofthe array. The preferred embodiment of this invention, furthermore,allows expression of the particle in an expression host guaranteeingproper folding and assembly of the VLP, to which the antigen, i.e theAβ1-6 peptide, is then further coupled

The present invention discloses methods of binding of Aβ1-6 peptide toVLPs. As indicated, in one aspect of the invention, the Aβ1-6 peptide isbound to the VLP by way of chemical cross-linking, typically andpreferably by using a heterobifunctional cross-linker. Severalhetero-bifunctional cross-linkers are known to the art. In preferredembodiments, the hetero-bifunctional cross-linker contains a functionalgroup which can react with preferred first attachment sites, i.e. withthe side-chain amino group of lysine residues of the VLP or at least oneVLP subunit, and a further functional group which can react with apreferred second attachment site, i.e. a cysteine residue fused to theAβ1-6 peptide and optionally also made available for reaction byreduction. The first step of the procedure, typically called thederivatization, is the reaction of the VLP with the cross-linker. Theproduct of this reaction is an activated VLP, also called activatedcarrier. In the second step, unreacted cross-linker is removed usingusual methods such as gel filtration or dialysis. In the third step, theAβ1-6 peptide is reacted with the activated VLP, and this step istypically called the coupling step. Unreacted Aβ1-6 peptide may beoptionally removed in a fourth step, for example by dialysis. Severalhetero-bifunctional cross-linkers are known to the art. These includethe preferred cross-linkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS,Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and othercross-linkers available for example from the Pierce Chemical Company(Rockford, Ill., USA), and having one functional group reactive towardsamino groups and one functional group reactive towards cysteineresidues. The above mentioned cross-linkers all lead to formation of athioether linkage. Another class of cross-linkers suitable in thepractice of the invention is characterized by the introduction of adisulfide linkage between the Aβ1-6 peptide and the VLP upon coupling.Preferred cross-linkers belonging to this class include for example SPDPand Sulfo-LC-SPDP (Pierce). The extent of derivatization of the VLP withcross-linker can be influenced by varying experimental conditions suchas the concentration of each of the reaction partners, the excess of onereagent over the other, the pH, the temperature and the ionic strength.The degree of coupling, i.e. the amount of Aβ1-6 peptides per subunitsof the VLP can be adjusted by varying the experimental conditionsdescribed above to match the requirements of the vaccine.

A particularly favored method of binding of Aβ1-6 peptides to the VLP,is the linking of a lysine residue on the surface of the VLP with acysteine residue on the Aβ1-6 peptide. In some embodiments, fusion of anamino acid linker containing a cysteine residue, as a second attachmentsite or as a part thereof, to Aβ1-6 for coupling to the VLP may berequired.

In general, flexible amino acid linkers are favored. Examples of theamino acid linker are selected from the group consisting of: (a) CGG;(b) N-terminal gamma 1-linker, (c) N-terminal gamma 3-linker; (d) Ighinge regions; (e) N-terminal glycine linkers; (f) (G)_(k)C(G), withn=0.12 and k=0-5 (SEQ ID NO: 34); (g) N-terminal glycine-serine linkers;(h) (G)_(k)C(G)_(m)(S)_(l)(GGGGS)_(n) with n=0-3, k=0-5, m=0-10, 1=0-2(SEQ ID NO: 35); (i) GGC; (k) KGGC-NH2; (l) C-terminal gamma l-linker;(m) C-terminal gamma 3-linker; (n) C-terminal glycine linkers; (O)(G)_(n)C(G)_(k) with n=0-12 and k=0-5 (SEQ ID NO: 36); (p) C-terminalglycine-serine linkers; (q) (G)_(m)(S)_(l)(GGGGS)_(n)(G)_(o)C(G)_(k)with n=0-3, k=0-5, m=0-10, l=0-2, and o=0-8 (SEQ ID NO: 37).

Further examples of amino acid linkers are the hinge region ofImmunoglobulins, glycine serine linkers (GGGGS). (SEQ ID NO: 38), andglycine linkers (G)_(n) all further containing a cysteine residue assecond attachment site and optionally further glycine residues.Typically preferred examples of said amino acid linkers are N-terminalgamma1: CGDKTHTSPP (SEQ ID NO: 39); C-terminal gamma 1: DKTHTSPPCG (SEQID NO: 40); N-terminal gamma 3: CGGPKPSTPPGSSGGAP (SEQ ID NO: 41);C-terminal gamma 3: PKPSTPPGSSGGAPGGCG (SEQ ID NO: 42); N-terminalglycine linker: GCGGGG (SEQ ID NO: 43) and C-terminal glycine linker:GGGGCG (SEQ ID NO: 44).

Other amino acid linkers particularly suitable in the practice of theinvention, when a hydrophobic Aβ peptide is bound to a VLP, are CGKKGG(SEQ ID NO: 46), or CGDEGG (SEQ ID NO: 31) for N-terminal linkers, orGGKKGC (SEQ ID NO: 45) and GGEDGC (SEQ ID NO: 32), for the C-terminallinkers. For the C-terminal linkers, the terminal cysteine is optionallyC-terminally amidated.

In preferred embodiments of the present invention, GGCG (SEQ ID NO: 47),GGC or GGC-NH2 (“NH2” stands for amidation) linkers at the C-terminus ofthe peptide or CGG at its N-terminus are preferred as amino acidlinkers. In general, glycine residues will be inserted between bulkyamino acids and the cysteine to be used as second attachment site, toavoid potential steric hindrance of the bulkier amino acid in thecoupling reaction. In the most preferred embodiment of the invention,the amino acid linker GGC-NH2 is fused to the C-terminus of Aβ1-6.

The cysteine residue present on the Aβ1-6 peptide has to be in itsreduced state to react with the hetero-bifunctional cross-linker on theactivated VLP, that is a free cysteine or a cysteine residue with a freesulfhydryl group has to be available. In the instance where the cysteineresidue to function as binding site is in an oxidized form, for exampleif it is forming a disulfide bridge, reduction of this disulfide bridgewith e.g. DTT, TCEP or β-mercaptoethanol is required. Low concentrationsof reducing agent are compatible with coupling as described in WO02/056905, higher concentrations inhibit the coupling reaction, as askilled artisan would know, in which case the reductand has to beremoved or its concentration decreased prior to coupling, e.g. bydialysis, gel filtration or reverse phase HPLC.

Binding of the Aβ1-6 peptide to the VLP by using a hetero-bifunctionalcross-linker according to the preferred methods described above, allowscoupling of Aβ1-6 peptide to the VLP in an oriented fashion. Othermethods of binding the Aβ1-6 peptide to the VLP include methods whereinthe Aβ1-6 peptide is cross-linked to the VLP using the carbodiimide EDC,and NHS. In further methods, the Aβ1-6 peptide is attached to the VLPusing a homo-bifunctional cross-linker such as glutaraldehyde, DSG,BM[PEO]₄, BS³, (Pierce Chemical Company, Rockford, Ill., USA) or otherknown homo-bifunctional cross-linkers with functional groups reactivetowards amine groups or carboxyl groups of the VLP.

Other methods of binding the VLP to a Aβ1-6 peptide include methodswhere the VLP is biotinylated, and the Aβ1-6 peptide expressed as astreptavidin-fusion protein, or methods wherein both the Aβ1-6 peptideand the VLP are biotinylated, for example as described in WO 00/23955.In this case, the Aβ1-6 peptide may be first bound to streptavidin oravidin by adjusting the ratio of Aβ1-6 peptide to streptavidin such thatfree binding sites are still available for binding of the VLP, which isadded in the next step. Alternatively, all components may be mixed in a“one pot” reaction. Other ligand-receptor pairs, where a soluble form ofthe receptor and of the ligand is available, and are capable of beingcross-linked to the VLP or the Aβ1-6 peptide, may be used as bindingagents for binding Aβ1-6 peptide to the VLP. Alternatively, either theligand or the receptor may be fused to the Aβ1-6 peptide, and so mediatebinding to the VLP chemically bound or fused either to the receptor, orthe ligand respectively. Fusion may also be effected by insertion orsubstitution.

As already indicated, in a favored embodiment of the present invention,the VLP is the VLP of a RNA phage, and in a more preferred embodiment,the VLP is the VLP of RNA phage Q coat protein.

One or several antigen molecules, i.e. a Aβ1-6 peptide, can be attachedto one subunit of the capsid or VLP of RNA phages coat proteins,preferably through the exposed lysine residues of the VLP of RNA phages,if sterically allowable. A specific feature of the VLP of the coatprotein of RNA phages and in particular of the Qβ coat protein VLP isthus the possibility to couple several antigens per subunit. This allowsfor the generation of a dense antigen array.

In a preferred embodiment of the invention, the binding and attachment,respectively, of the at least one Aβ1-6 peptide to the virus-likeparticle is by way of interaction and association, respectively, betweenat least one first attachment site of the virus-like particle and atleast one second attachment of the antigen or antigenic determinant.

VLPs or capsids of Qβ coat protein display a defined number of lysineresidues on their surface, with a defined topology with three lysineresidues pointing towards the interior of the capsid and interactingwith the RNA, and four other lysine residues exposed to the exterior ofthe capsid. These defined properties favor the attachment of antigens tothe exterior of the particle, rather than to the interior of theparticle where the lysine residues interact with RNA. VLPs of other RNAphage coat proteins also have a defined number of lysine residues ontheir surface and a defined topology of these lysine residues.

In further preferred embodiments of the present invention, the firstattachment site is a lysine residue and/or the second attachmentcomprises sulfhydryl group or a cysteine residue. In a very preferredembodiment of the present invention, the first attachment site is alysine residue and the second attachment is a cysteine residue.

In very preferred embodiments of the invention, the Aβ1-6 peptide isbound via a cysteine residue, to lysine residues of the VLP of RNA phagecoat protein, and in particular to the VLP of Qβ coat protein.

Another advantage of the VLPs derived from RNA phages is their highexpression yield in bacteria that allows production of large quantitiesof material at affordable cost.

As indicated, the inventive conjugates and arrays, respectively, differfrom prior art conjugates in their highly organized structure,dimensions, and in the repetitiveness of the antigen on the surface ofthe array. Moreover, the use of the VLPs as carriers allow the formationof robust antigen arrays and conjugates, respectively, with variableantigen density. In particular, the use of VLPs of RNA phages, andhereby in particular the use of the VLP of RNA phage Qβ coat proteinallows achieving very high epitope density. In particular, a density ofmore than 1.5 epitopes per subunit could be reached by coupling thehuman Aβ1-6 peptide to the VLP of Qβ coat protein. The preparation ofcompositions of VLPs of RNA phage coat proteins with a high epitopedensity can be effected using the teaching of this application. Inpreferred embodiment of the invention, when a Aβ1-6 peptide is coupledto the VLP of Qβ3 coat protein, an average number of Aβ1-6 peptide persubunit of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 0.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 2.5, 2.6, 2.7, 2.8, 2.9, orhigher is preferred.

The second attachment site, as defined herein, may be either naturallyor non-naturally present with the antigen or the antigenic determinant.In the case of the absence of a suitable natural occurring secondattachment site on the antigen or antigenic determinant, such a, thennon-natural second attachment has to be engineered to the antigen.

As described above, four lysine residues are exposed on the surface ofthe VIP of Qβ coat protein. Typically these residues are derivatizedupon reaction with a cross-linker molecule. In the instance where notall of the exposed lysine residues can be coupled to an antigen, thelysine residues which have reacted with the cross-linker are left with across-linker molecule attached to the ε-amino group after thederivatization step. This leads to disappearance of one or severalpositive charges, which may be detrimental to the solubility andstability of the VLP. By replacing some of the lysine residues witharginines, as in the disclosed Qβ coat protein mutants described below,we prevent the excessive disappearance of positive charges since thearginine residues do not react with the cross-linker. Moreover,replacement of lysine residues by arginines may lead to more definedantigen arrays, as fewer sites are available for reaction to theantigen.

Accordingly, exposed lysine residues were replaced by arginines in thefollowing Qβ coat protein mutants and mutant Qβ VLPs disclosed in thisapplication: Qβ-240 (Lys13-Arg; SEQ ID NO:17), Qβ-250 (Lys 2-Arg,Lys13-Arg; SEQ ID NO: 19) and Qβ-259 (Lys 2-Arg, Lys16-Arg; SEQ IDNO:21). The constructs were cloned, the proteins expressed, the VLPspurified and used for coupling to peptide and protein antigens. Qβ-251;(SEQ ID NO: 20 was also constructed, and guidance on how to express,purify and couple the VLP of Qβ-251 coat protein can be found throughoutthe application.

In a further embodiment, we disclose a Qβ mutant coat protein with oneadditional lysine residue, suitable for obtaining even higher densityarrays of antigens. This mutant Qβ coat protein, Qβ-243 (Asn 10-Lys; SEQID NO: 18), was cloned, the protein expressed, and the capsid or VLPisolated and purified, showing that introduction of the additionallysine residue is compatible with self-assembly of the subunits to acapsid or VLP. Thus, Aβ1-6 peptide arrays and conjugates, respectively,may be prepared using VLP of Qβ coat protein mutants. A particularlyfavored method of attachment of antigens to VLPs, and in particular toVLPs of RNA phage coat proteins is the linking of a lysine residuepresent on the surface of the VIP of RNA phage coat proteins with acysteine residue added to the antigen, i.e. the Aβ1-6 peptide. In orderfor a cysteine residue to be effective as second attachment site, asulthydryl group must be available for coupling. Thus, a cysteineresidue has to be in its reduced state, that is, a free cysteine or acysteine residue with a free sulfhydryl group has to be available. Inthe instant where the cysteine residue to function as second attachmentsite is in an oxidized form, for example if it is forming a disulfidebridge, reduction of this disulfide bridge with e.g. DT, TCEP orβ-mercaptoethanol is required. The concentration of reductand, and themolar excess of reductand over antigen has to be adjusted for eachantigen. A titration range, starting from concentrations as low as 10 μMor lower, up to 10 to 20 mM or higher reductand if required is tested,and coupling of the antigen to the carrier assessed. Although lowconcentrations of reductand are compatible with the coupling reaction asdescribed in WO 02/056905, higher concentrations inhibit the couplingreaction, as a skilled artisan would know, in which case the reductandhas to be removed or its concentration decreased, e.g. by dialysis, gelfiltration or reverse phase HPLC. Advantageously, the pH of the dialysisor equilibration buffer is lower than 7, preferably 6. The compatibilityof the low pH buffer with antigen activity or stability has to betested.

Epitope density on the VLP of RNA phage coat proteins can be modulatedby the choice of cross-linker and other reaction conditions. Forexample, the cross-linkers Sulfo-GMBS and SMPH typically allow reachinghigh epitope density. Derivatization is positively influenced by highconcentration of reactands, and manipulation of the reaction conditionscan be used to control the number of antigens coupled to VLPs of RNAphage coat proteins, and in particular to VLPs of Qβ coat protein.

Prior to the design of a non-natural second attachment site the positionat which it should be fused, inserted or generally engineered has to bechosen. The selection of the position of the second attachment site may,by way of example, be based on a crystal structure of the antigen. Sucha crystal structure of the antigen may provide information on theavailability of the C- or N-termini of the molecule (determined forexample from their accessibility to solvent), or on the exposure tosolvent of residues suitable for use as second attachment sites, such ascysteine residues. Exposed disulfide bridges, as is the case for Fabfragments, may also be a source of a second attachment site, since theycan be generally converted to single cysteine residues through mildreduction, with e.g. 2-mercaptoethylamine, TCEP, β-mercaptoethanol orDTT. Mild reduction conditions not affecting the immunogenicity of theantigen will be chosen. In general, in the case where immunization witha self-antigen is aiming at inhibiting the interaction of thisself-antigen with its natural ligands, the second attachment site willbe added such that it allows generation of antibodies against the siteof interaction with the natural ligands. Thus, the location of thesecond attachment site will be selected such that steric hindrance fromthe second attachment site or any amino acid linker containing the sameis avoided. In further embodiments, an antibody response directed at asite distinct from the interaction site of the self-antigen with itsnatural ligand is desired. In such embodiments, the second attachmentsite may be selected such that it prevents generation of antibodiesagainst the interaction site of the self-antigen with its naturalligands.

Other criteria in selecting the position of the second attachment siteinclude the oligomerization state of the antigen, the site ofoligomerization, the presence of a cofactor, and the availability ofexperimental evidence disclosing sites in the antigen structure andsequence where modification of the antigen is compatible with thefunction of the self-antigen, or with the generation of antibodiesrecognizing the self-antigen.

In the most preferred embodiments, the Aβ1-6 peptide comprises a singlesecond attachment site or a single reactive attachment site capable ofassociation with the first attachment sites on the core particle and theVLPs or VIP subunits, respectively. This ensures a defined and uniformbinding and association, respectively, of the at least one, buttypically more than one, preferably more than 10, 20, 40, 80, 120, 150,180, 210, 240, 270, 300, 360, 400, 450 antigens to the core particle andVLP, respectively. The provision of a single second attachment site or asingle reactive attachment site on the antigen, thus, ensures a singleand uniform type of binding and association, respectively leading to avery highly ordered and repetitive array. For example, if the bindingand association, respectively, is effected by way of a lysine- (as thefirst attachment site) and cysteine- (as a second attachment site)interaction, it is ensured, in accordance with this preferred embodimentof the invention, that only one cysteine residue per antigen,independent whether this cysteine residue is naturally or non-naturallypresent on the antigen, is capable of binding and associating,respectively, with the VLP and the first attachment site of the coreparticle, respectively.

In some embodiments, engineering of a second attachment site onto theantigen require the fusion of an amino acid linker containing an aminoacid suitable as second attachment site according to the disclosures ofthis invention. Therefore, in a preferred embodiment of the presentinvention, an amino acid linker is bound to the antigen or the antigenicdeterminant by way of at least one covalent bond. Preferably, the aminoacid linker comprises, or alternatively consists of, the secondattachment site. In a further preferred embodiment, the amino acidlinker comprises a sulfhydryl group or a cysteine residue. In anotherpreferred embodiment, the amino acid linker is cysteine. Some criteriaof selection of the amino acid linker as well as further preferredembodiments of the amino acid linker according to the invention havealready been mentioned above.

In a further preferred embodiment of the invention, the at least oneantigen or antigenic determinant, i.e. the Aβ1-6 peptide is fused to thevirus-like particle. As outlined above, a VLP is typically composed ofat least one subunit assembling into a VLP. Thus, in again a furtherpreferred embodiment of the invention, the antigen or antigenicdeterminant, preferably the at least one Aβ1-6 peptide is fused to atleast one subunit of the virus-like particle or of a protein capable ofbeing incorporated into a VLP generating a chimeric VLP-subunit-Aβ1-6peptide protein fusion.

Fusion of the Aβ1-6 peptides can be effected by insertion into the VLPsubunit sequence, or by fusion to either the N- or C-terminus of theVLP-subunit or protein capable of being incorporated into a VLP.Hereinafter, when referring to fusion proteins of a peptide to a VLPsubunit, the fusion to either ends of the subunit sequence or internalinsertion of the peptide within the subunit sequence are encompassed.

Fusion may also be effected by inserting the Aβ1-6 peptide sequencesinto a variant of a VLP subunit where part of the subunit sequence hasbeen deleted, that are further referred to as truncation mutants.Truncation mutants may have N- or C-terminal, or internal deletions ofpart of the sequence of the VLP subunit. For example, the specific VLPHBcAg with, for example, deletion of amino acid residues 79 to 81 is atruncation mutant with an internal deletion. Fusion of Aβ1-6 peptides toeither the N- or C-terminus of the truncation mutants VLP-subunits alsolead to embodiments of the invention. Likewise, fusion of an epitopeinto the sequence of the VLP subunit may also be effected bysubstitution, where for example for the specific VLP HBcAg, amino acids79-81 are replaced with a foreign epitope. Thus, fusion, as referred tohereinafter, may be effected by insertion of the Aβ1-6 peptide sequencein the sequence of a VLP subunit, by substitution of part of thesequence of the VLP subunit with the Aβ1-6 peptide, or by a combinationof deletion, substitution or insertions.

The chimeric Aβ1-6 peptide-VLP subunit will be in general capable ofself-assembly into a VLP. VLP displaying epitopes fused to theirsubunits are also herein referred to as chimeric VLPs. As indicated, thevirus-like particle comprises or alternatively is composed of at leastone VLP subunit. In a further embodiment of the invention, thevirus-like particle comprises or alternatively is composed of a mixtureof chimeric VLP subunits and non-chimeric VLP subunits, i.e. VLPsubunits not having an antigen fused thereto, leading to so calledmosaic particles. This may be advantageous to ensure formation of, andassembly to a VLP. In those embodiments, the proportion of chimericVLP-subunits may be 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% orhigher.

Flanking amino acid residues may be added to either end of the sequenceof the peptide or epitope to be fused to either end of the sequence ofthe subunit of a VLP, or for internal insertion of such peptidicsequence into the sequence of the subunit of a VLP. Glycine and serineresidues are particularly favored amino acids to be used in the flankingsequences added to the peptide to be fused. Glycine residues conferadditional flexibility, which may diminish the potentially destabilizingeffect of fusing a foreign sequence into the sequence of a VLP subunit.

In a specific embodiment of the invention, the VLP is a Hepatitis B coreantigen VLP. Fusion proteins of the Aβ1-6 peptide to either theN-terminus of a HBcAg (Neyrinck, S. et al., Nature Med. 5:1157-1163(1999)) or insertions in the so called major immunodominant region (MIR)have been described (Pumpens, P. and Grens, E., Intervirology 44:98-114(2001)), WO 01/98333), and are preferred embodiments of the invention.Naturally occurring variants of HBcAg with deletions in the MIR havealso been described (Pumpens, P. and Grens, E., Intervirology 44:98-114(2001), which is expressly incorporated by reference in its entirety),and fusions to the N- or C-terminus, as well as insertions at theposition of the MIR corresponding to the site of deletion as compared toa wt HBcAg are further embodiments of the invention. Fusions to theC-terminus have also been described (Pumpens, P. and Grens, E.,Intervirology 44:98-114 (2001)). One skilled in the art will easily findguidance on how to construct fusion proteins using classical molecularbiology techniques (Sambrook, J. et al., eds., Molecular Cloning, ALaboratory Manual, 2nd. edition, Cold Spring Habor Laboratory Press,Cold Spring Harbor, N.Y. (1989), Ho et al., Gene 77:51 (1989)). Vectorsand plasmids encoding HBcAg and HBcAg fusion proteins and useful for theexpression of a HBcAg and HBcAg fusion proteins have been described(Pumpens, P. & Grens, E. Intervirology 44: 98-114 (2001), Neyrinck, S.et al., Nature Med. 5:1157-1163 (1999)) and can be used in the practiceof the invention. We also describe by way of example (Example 6) theinsertion of an epitope into the MIR of HBcAg, resulting in a chimericself-assembling HBcAg. An important factor for the optimization of theefficiency of self-assembly and of the display of the epitope to beinserted in the MIR of HBcAg is the choice of the insertion site, aswell as the number of amino acids to be deleted from the HBcAg sequencewithin the MIR (Pumpens, P. and Grens, E., Intervirology 44:98-114(2001); EP 0 421 635; U.S. Pat. No. 6,231,864) upon insertion, or inother words, which amino acids form HBcAg are to be substituted with thenew epitope. For example, substitution of HBcAg amino acids 76-80,79-81, 79-80, 75-85 or 80-81 with foreign epitopes has been described(Pumpens, P. and Grens, E., Intervirology 44:98-114 (2001); EP0421635;U.S. Pat. No. 6,231,864). HBcAg contains a long arginine tail (Pumpens,P. and Grens, E., Intervirology 44:98-114 (2001)) which is dispensablefor capsid assembly and capable of binding nucleic acids (Pumpens, P.and Grens, E., Intervirology 44:98-114 (2001)). HBcAg either comprisingor lacking this arginine tail are both embodiments of the invention.

In a further preferred embodiment of the invention, the VLP is a VLP ofa RNA phage. The major coat proteins of RNA phages spontaneouslyassemble into VLPs upon expression in bacteria, and in particular in E.coli. Specific examples of bacteriophage coat proteins which can be usedto prepare compositions of the invention include the coat proteins ofRNA bacteriophages such as bacteriophage Qβ (SEQ ID NO:4; PIR Database,Accession No. VCBPQβ referring to Qβ CP and SEQ ID NO: 5; Accession No.AAA16663 referring to Qβ A1 protein) and bacteriophage fr (SEQ ID NO: 7;PIR Accession No. VCBPFR).

In a more preferred embodiment, the at least one Aβ1-6 peptide is fusedto a Qβ coat protein. Fusion protein constructs wherein epitopes havebeen fused to the C-terminus of a truncated form of the A1 protein ofQβ, or inserted within the A1 protein have been described (Kozlovska, T.M. et al., Intervirology, 39:9-15 (1996)). The A1 protein is generatedby suppression at the UGA stop codon and has a length of 329 aa, or 328aa, if the cleavage of the N-terminal methionine is taken into account.Cleavage of the N-terminal methionine before an alanine (the secondamino acid encoded by the Qβ CP gene) usually takes place in E. coli,and such is the case for N-termini of the Qβ coat proteins. The part ofthe A1 gene, 3′ of the UGA amber codon encodes the CP extension, whichhas a length of 195 amino acids. Insertion of the at least one Aβ1-6peptide between position 72 and 73 of the CP extension leads to furtherembodiments of the invention (Kozlovska, T. M., et al., Intervirology39:9-15 (1996)). Fusion of a Aβ1-6 peptide at the C-terminus of aC-terminally truncated Qβ A1 protein leads to further preferredembodiments of the invention. For example, Kozlovska et al.,(Intervirology, 39: 9-15 (1996)) describe Qβ A1 protein fusions wherethe epitope is fused at the C-terminus of the Qβ CP extension truncatedat position 19.

As described by Kozlovska et al. (Intervirology, 39: 9-15 (1996)),assembly of the particles displaying the fused epitopes typicallyrequires the presence of both the A1 protein-Aβ1-6 peptide fusion andthe wt CP to form a mosaic particle. However, embodiments comprisingvirus-like particles, and hereby in particular the VLPs of the RNA phageQβ coat protein, which are exclusively composed of VLP subunits havingat least one Aβ1-6 peptide fused thereto, are also within the scope ofthe present invention.

The production of mosaic particles may be effected in a number of ways.Kozlovska et al., Intervirology, 39:9-15 (1996), describe three methods,which all can be used in the practice of the invention. In the firstapproach, efficient display of the fused epitope on the VLPs is mediatedby the expression of the plasmid encoding the Qβ A1 protein fusionhaving a UGA stop codong between CP and CP extension in a E. coli strainharboring a plasmid encoding a cloned UGA suppressor tRNA which leads totranslation of the UGA codon into Trp (pISM3001 plasmid (Smiley B. K.,et al., Gene 134:33-40 (1993))). In another approach, the CP gene stopcodon is modified into UAA, and a second plasmid expressing the A1protein-Aβ1-6 peptide fusion is cotransformed. The second plasmidencodes a different antibiotic resistance and the origin of replicationis compatible with the first plasmid (Kozlovska, T. M., et al.,Intervirology 39:9-15 (1996)). In a third approach, CP and the A1protein-Aβ1-6 peptide fusion are encoded in a bicistronic manner,operatively linked to a promoter such as the Trp promoter, as describedin FIG. 1 of Kozlovska et al., Intervirology, 39:9-15 (1996).

In a further embodiment, the Aβ1-6 peptide is inserted between aminoacid 2 and 3 (numbering of the cleaved CP, that is wherein theN-terminal methionine is cleaved) of the fr CP, thus leading to a Aβ 1-6peptide-fr CP fusion protein. Vectors and expression systems forconstruction and expression of fr CP fusion proteins self-assembling toVLP and useful in the practice of the invention have been described(Pushko P. et al., Prot. Eng. 6:883-891 (1993)). In a specificembodiment, the Aβ1-6 peptide sequence is inserted into a deletionvariant of the fr CP after amino acid 2, wherein residues 3 and 4 of thefr CP have been deleted (Pushko P. et al., Prot. Eng. 6:883-891 (1993)).

Fusion of epitopes in the N-terminal protuberant β-hairpin of the coatprotein of RNA phage MS-2 and subsequent presentation of the fusedepitope on the self-assembled VLP of RNA phage MS-2 has also beendescribed (WO 92/13081), and fusion of Aβ1-6 peptide by insertion orsubstitution into the coat protein of MS-2 RNA phage is also fallingunder the scope of the invention.

In another embodiment of the invention, the Aβ1-6 peptide is fused to acapsid protein of papillomavirus. In a more specific embodiment, theAβ1-6 peptide is fused to the major capsid protein L1 of bovinepapillomavirus type 1 (BPV-1). Vectors and expression systems forconstruction and expression of BPV-1 fusion proteins in abaculovirus/insect cells systems have been described (Chackerian, B. etal., Proc. Nail. Acad. Sci. USA 96:2373-2378 (1999), WO 00/23955).Substitution of amino acids 130-136 of BPV-1 L1 with a Aβ1-6 peptideleads to a BPV-1 L1-A1-6 peptide fusion protein, which is a preferredembodiment of the invention, Cloning in a baculovirus vector andexpression in baculovirus infected Sf9 cells has been described, and canbe used in the practice of the invention (Chackerian, B. et al., Proc.Natl. Acad. Sci. USA 96:2373-2378 (1999), WO 00/23955). Purification ofthe assembled particles displaying the fused Aβ1-6 peptide can beperformed in a number of ways, such as for example gel filtration orsucrose gradient ultracentrifugation (Chackerian, B. et al., Proc. Natl.Acad. Sci. USA 96:2373-2378 (1999), WO 00/23955).

In a further embodiment of the invention, the Aβ1-6 peptide is fused toa Ty protein capable of being incorporated into a Ty VLP. In a morespecific embodiment, the Aβ1-6 peptide is fused to the p1 or capsidprotein encoded by the TYA gene (Roth, J. F., Yeast 16:785-795 (2000)).The yeast retrotransposons Ty1, 2, 3 and 4 have been isolated fromSaccharomyces Serevisiae, while the retrotransposon Tf1 has beenisolated from Schizosaccharomyces Pombae (Boeke, J. D. and Sandmeyer, S.B., “Yeast Transposable elements,” in The molecular and Cellular Biologyof the Yeast Saccharomyces: Genome dynamics, Protein Synthess, andEnergetics, p. 193, Cold Spring Harbor Laboratory Press (1991)). Theretrotransposons Ty1 and 2 are related to the copia class of plant andanimal elements, while Ty3 belongs to the gypsy family ofretrotransposons, which is related to plants and animal retroviruses. Inthe Ty1 retrotransposon, the p1 protein, also referred to as Gag orcapsid protein, has a length of 440 amino acids. P1 is cleaved duringmaturation of the VLP at position 408, leading to the p2 protein, theessential component of the VLP.

Fusion proteins to p1 and vectors for the expression of said fusionproteins in Yeast have been described (Adams, S. E., et al., Nature329:68-70 (1987)). So, for example, a Aβ1-6 peptide may be fused to p1by inserting a sequence coding for the Aβ1-6 peptide into the BamH1 siteof the pMA5620 plasmid (Adams, S. E., et al, Nature 329:68-70 (1987)).The cloning of sequences coding for foreign epitopes into the pMAS620vector leads to expression of fusion proteins comprising amino acids1-381 of p1 of Ty1-15, fused C-terminally to the N-terminus of theforeign epitope. Likewise, N-terminal fusion of a Aβ1-6 peptide, orinternal insertion into the p1 sequence, or substitution of part of thep1 sequence are also meant to fall within the scope of the invention. Inparticular, insertion of a Aβ1-6 peptide into the Ty sequence betweenamino acids 30-31, 67-68, 113-114 and 132-133 of the Ty protein p1(EP0677111) leads to preferred embodiments of the invention.

Further VLPs suitable for fusion of Aβ1-6 peptides are, for example,Retrovirus-like-particles (WO9630523), HIV2 Gag (Kang, Y. C., et al,Biol. Chem. 380:353-364 (1999)), Cowpea Mosaic Virus (Taylor, K. M. etal. Biol. Chem. 380:387-392 (1999)), parvovirus VP2 VLP (Rueda, P. etal., Virology 263:89-99 (1999)), HBsAg (U.S. Pat. No. 4,722,840,EP002041681).

Examples of chimeric VLPs suitable for the practice of the invention armalso those described in Intervirology 39:1 (1996). Further examples ofVLPs contemplated for use in the invention are: HPV-1, HPV-6, HPV-1,HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV, HIV GAG, Tobacco MosaicVirus. Virus-like particles of SV-40, Polyomavirus, Adenovirus, HerpesSimplex Virus, Rotavirus and Norwalk virus have also been made, andchimeric VLPs of those VLPs comprising a Aβ1-6 peptide are also withinthe scope of the present invention.

In preferred embodiments of the invention, Aβ1-6 peptides suitable forgenerating vaccines of the invention are modified with an amino acidlinker for binding to a VLP. Those Aβ1-6 peptides include, but are notlimited to: Aβ1-6 fused C-terminally to the linker GGC. Amino acidlinkers suitable for fusion to the N-terminus of Aβ1-6 fragments includebut are not limited to the sequence COG and CGHGNKS. Linkers suitablefor fusion to the C-terminus of Aβ1-6 include but are not limited to thesequence GGC. In a preferred embodiment, when a linker is fused to theC-terminus of Aβ or Aβ fragments, the C-terminal Cysteine is amidated.In a preferred embodiment, Aβ1-6 is fused to an amino acid linker andhas the sequence: “NH2-DAEFRHGGC-CONH2, wherein the C-terminal Cysteineis amidated, which is indicated by the C-terminal “—CONH2”, and theN-terminus of the peptide is free, which is further indicated by “NH2-”.Amino acid linkers are preferably short, to avoid induction of immuneresponses against amino acids of said linker, but should allow theinduction of antibodies cross-reactive with soluble Aβ and AD plaquesand may facilitate the interaction of antibodies with the Aβ1-6 peptide.Other suitable properties of the amino acid linker are flexibility, andpreferably lack of bulky amino acids which might interfere withcoupling, and/or generate an immune response against the linker itself.In more preferred embodiments, the amino acid linker containing acysteine residue as second attachment site is fused to the C-terminus ofthe Aβ1-6 peptide.

Additional Aβ fragments suitable in the practice of the inventioninclude Aβ fragments corresponding to the aforementioned fragments, alsomodified as described above, from other animal species and elicitingAntibodies cross-reactive with human amyloid plaques and soluble humanAβ. Examples of such fragments are Aβ1-6 from primates (DAEFRH: SEQ IDNO: 84), rabbit (DAEFRH; SEQ ID NO: 85), guinea pig (DAEFRH: SEQ ID NO:88), mouse (DAEFGH; SEQ ID) NO: 76), rat (DAEFGH SEQ ID NO: 87), andxaenopus laevis (DSEYRH; 86).

A number of animal models of AD based on transgenic mice overexpressingmutated forms of human APP have been reported (Games, D. et al., Nature373: 523-527 (1995a); Sturchler-Pierrat et al., Proc. Nail. Acad. Sci.USA 94: 13287-13292 (1997); Hsiao, K., et al., Science 274: 99-102(1996); Chen, G. et al., Nature 408: 975-979 (2000); Janus, C. et al.,Nature 408: 979-982 (2000); Morgan, D. et al., Nature 408: 982-985(2000)). Those mice models differ from each other in the level ofoverexpression of the transgene, the AD mutations present on thetransgene and the promoter under which overexpression of the transgeneis directed. These animal models fail to display all of the pathologicalsigns of AD, which are in particular age-related changes in behaviour,deposition of β-amyloid into insoluble plaques, neurofibrillary tangleswithin neurons, and loss of neurons throughout the forebrain (Chapman,P. F. Nature 408: 915-916 (2000)). Memory deficits and methods toidentify them could however be identified in those models, and may beused in testing the effect of the compositions of the invention inanimal models (Chen, G. et al., Nature 408: 975-979 (2000); Janus, C. etal., Nature 408: 979-982 (2000); Morgan, D. et al., Nature 408: 982-985(2000)). Furthermore, age related deposition of Aβ into amyloid plaquescan be studied in those models, which also develop astrocytosis andmicrogliosis.

It will be understood by one of ordinary skill in the relevant arts thatother suitable modifications and adaptations to the methods andapplications described herein are readily apparent and may be madewithout departing from the scope of the invention or any embodimentthereof. Having now described the present invention in detail, the samewill be more clearly understood by reference to the following examples,which are included herewith for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES Example 1 Cloning and Construction, Respectively, Expressionand Purification of Preferred Core Particles and VLP of RNA Phages,Respectively A. Construction and Expression of Mutant Qβ Coat Proteins,and Purification of Mutant Qβ Coat Protein VLPs or Capsids PlasmidConstruction and Cloning of Mutant Coat Proteins

Construction of pQβ-240:

The plasmid pQβ10 (Kozlovska, T M, et al., Gene 137:133-137) was used asan initial plasmid for the construction of pQβ-240. The mutationLys13→Arg was created by inverse PCR. The inverse primers were designedin inverted tail-to-tail directions:

(SEQ ID NO: 48) 5′-GGTAACATGGTCGAGATGGAAAACAAACTCTGGTCC-3′ and(SEQ ID No: 49) 5′-GGACCAGAGTTTGTTTTCCATCTCGACCGATGTTACC-3′.

The products of the first PCR were used as templates for the second PCRreaction, in which an upstream primer

(SEQ ID NO: 50) 5′-AGCTCGCCCGGGGATCCTCTAG-3′ and a downstream primer(SEQ ID NO: 51) 5′-CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3′

-   -   were used. The product of the second PCR was digested with Xba1        and Mph1103I and cloned into the pQβ10 expression vector, which        was cleaved by the same restriction enzymes. The PCR reactions        were performed with PCR kit reagents and according to producer        protocol (MBI Fermentas, Vilnius, Lithuania).

Sequencing using the direct label incorporation method verified thedesired mutations. E. coli cells harbouring pQβ-240 supported efficientsynthesis of 14-kD protein co migrating upon SDS-PAGE with control Qβcoat protein isolated from Qβ phage particles.

Resulting amino acid sequence:  (SEQ ID NO: 17)AKLETVTLGNIGRDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQKYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

Construction of pQβ-243:

The plasmid pQβ10 was used as an initial plasmid for the construction ofpQβ-243. The mutation Asn10→Lys was created by inverse PCR. The inverseprimers were designed in inverted tail-to-tail directions:

(SEQ ID NO: 52) 5′-GGCAAAATTAGAGACTGTTACTTTAGGTAAGATCGG-3′ and(SEQ ID NO: 53) 5′-CCGATCTTACCTAAAGTAACAGTCTCTAATTTTGCC-3′.

The products of the first PCR were used as templates for the second PCRreaction, in which an upstream primer

(SEQ ID NO: 50) 5′-AGCTCGCCCGGGGATCCTCTAG-3′ and a downstream primer(SEQ ID NO: 51) 5′-CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3′

-   -   were used. The product of the second PCR was digested with XbaI        and Mph1103I and cloned into the pQβ10 expression vector, which        was cleaved by the same restriction enzymes. The PCR reactions        were performed with PCR kit reagents and according to producer        protocol (MBI Fermentas, Vilnius, Lithuania).

Sequencing using the direct label incorporation method verified thedesired mutations. E. coli cells harbouring pQβ-243 supported efficientsynthesis of 14-kD protein co migrating upon SDSD-PAGE with control Qβcoat protein isolated from Qβ phage particles.

Resulting amino acid sequence:  (SEQ ID NO: 18)AKLETVTLGKIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTQKYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

Construction of pQβ-250:

The plasmid pQβ-240 was used as an initial plasmid for the constructionof pQβ-250. The mutation Lys2→Arg was created by site-directedmutagenesis. An upstream primer

(SEQ ID NO: 54) 5′-GGCCATGGCACGACTCGAGACTGTTACTTTAGG-3′and a downstream primer (SEQ ID NO: 55) 5′-GATTTAGGTGACACTATAG-3′

were used for the synthesis of the mutant PCR-fragment, which wasintroduced into the pQβ-185 expression vector at the unique restrictionsites NcoI and HindIII. The PCR reactions were performed with PCR kitreagents and according to producer protocol (MBI Fermentas, Vilnius,Lithuania).

Sequencing using the direct label incorporation method verified thedesired mutations. E. coli cells harbouring pQβ-250 supported efficientsynthesis of 14-kD protein co migrating upon PAGE with control Qβ coatprotein isolated from Qβ phage particles.

Resulting amino acid sequence:  (SEQ ID NO: 19)ARLETVTLGNIGRDGKQTLVLNPRGVNPTNGVASLSQAGVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQKYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

Construction of pQβ-251:

The plasmid pQβ10 was used as an initial plasmid for the construction ofpQβ-251. The mutation Lys16→Arg was created by inverse PCR. The inverseprimers were designed in inverted tail-to-tail directions:

(SEQ ID NO: 56) 5′-GATGGACGTCAAACTCTGGTCCTCAATCCGCGTGGGG-3′ and(SEQ ID NO: 57) 5′-CCCCACGCGGATTGAGGACCAGAGTTTGACGTCCATC-3′

The products of the first PCR were used as templates for the second PCRreaction, in which an upstream primer

(SEQ ID NO: 50) 5′-AGCTCGCCCGGGGATCCTCTAG-3′ and a downstream primer(SEQ ID NO: 51) 5′-CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3′

were used. The product of the second PCR was digested with XbaI andMph1103I and cloned into the pQβ10 expression vector, which was cleavedby the same restriction enzymes. The PCR reactions were performed withPCR kit reagents and according to producer protocol (MBI Fermentas,Vilnius, Lithuania).

Sequencing using the direct label incorporation method verified thedesired mutations. E. coli cells harbouring pQβ-251 supported efficientsynthesis of 14-kD protein co migrating upon SDS-PAGE with control Qβcoat protein isolated from Qβ phage particles. The resulting amino acidsequence encoded by this construct is shown in (SEQ. ID NO: 20).

Construction of pQβ-259:

The plasmid pQβ-251 was used as an initial plasmid for the constructionof pQβ-259. The mutation Lys2→Arg was created by site-directedmutagenesis. An upstream primer

(SEQ ID NO: 54) 5′-GGCCATGGCACGACTCGAGACTGTTACTTTAGG-3′and a downstream primer (SEQ ID NO: 55) 5′-GATTTAGGTGACACTATAG-3′were used for the synthesis of the mutant PCR-fragment, which wasintroduced into the pQβ-185 expression vector at the unique restrictionsites NcoI and HindIII. The PCR reactions were performed with PCR kitreagents and according to producer protocol (MBI Fermentas, Vilnius,Lithuania).

Sequencing using the direct label incorporation method verified thedesired mutations. E. coli cells harbouring pQβ-259 supported efficientsynthesis of 14-kD protein co migrating upon SDS-PAGE with control Qβcoat protein isolated from Qβ phage particles.

Resulting amino acid sequence:  (SEQ ID NO: 21AKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQKYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

General Procedures for Expression and Purification of Qβ and Qβ Mutants

Expression

E. coli JM109 was transformed with Qβ coat protein expression plasmids.5 ml of LB liquid medium containing 20 μg/ml ampicillin were inoculatedwith clones transformed with Qβ coat protein expression plasmids. Theinoculated culture was incubated at 37° C. for 16-24 h without shaking.The prepared inoculum was subsequently diluted 1:100 in 100-300 ml offresh LB medium, containing 20 μg/ml ampicillin, and incubated at 37° C.overnight without shaking. The resulting second inoculum was diluted1:50 in M9 medium containing 1% Casamino acids and 0.2% glucose inflasks, and incubated at 37° C. overnight under shaking.

Purification

Solutions and buffers for the purification procedure:1. Lysis buffer LB50 mM Tris-HCl pH8.0 with 5 mM EDTA, 0.1% tritonX100 and freshlyprepared PMSF at a concentration of 5 micrograms per ml. Withoutlysozyme and DNAse.

2. SAS

-   -   Saturated ammonium sulphate in water

3. Buffer NT.

-   -   20 mM Tris-HCl, pH 7.8 with 5 mM EDTA and 150 mM NaCl.

4. PEG

-   -   40% (w/v) polyethylenglycol 6000 in NET

Disruption and Lysis

Frozen cells were resuspended in LB at 2 ml/g cells. The mixture wassonicated with 22 kH five times for 15 seconds, with intervals of 1 minto cool the solution on ice. The lysate was then centrifuged at 14 000rpm, for 1 h using a Janecki K 60 rotor. The centrifugation stepsdescribed below were all performed using the same rotor, exceptotherwise stated. The supernatant was stored at 4° C., while cell debriswere washed twice with LB. After centrifugation, the supernatants of thelysate and wash fractions were pooled.

Fractionation

A saturated ammonium sulphate solution was added dropwise under stirringto the above pooled lysate. The volume of the SAS was adjusted to be onefifth of total volume, to obtain 20% of saturation. The solution wasleft standing overnight, and was centrifuged the next day at 14 000 rpm,for 20 min. The pellet was washed with a small amount of 20% ammoniumsulphate, and centrifuged again. The obtained supernatants were pooled,and SAS was added dropwise to obtain 40% of saturation. The solution wasleft standing overnight, and was centrifuged the next day at 14 000 rpm,for 20 min. The obtained pellet was solubilised in NET buffer.

Chromatography

The capsid or VLP protein resolubilized in NET buffer was loaded on aSepharose CL-4B column. Three peaks eluted during chromatography. Thefirst one mainly contained membranes and membrane fragments, and was notcollected. Capsids were contained in the second peak, while the thirdone contained other E. coli proteins.

The peak fractions were pooled, and the NaCl concentration was adjustedto a final concentration of 0.65 M. A volume of PEG solutioncorresponding to one half of the pooled peak fraction was added dropwiseunder stirring. The solution was left to stand overnight withoutstirring. The capsid protein was sedimented by centrifugation at 14 000rpm for 20 min. It was then solubilized in a minimal volume of NET andloaded again on the Sepharose CL-413 column. The peak fractions werepooled, and precipitated with ammonium sulphate at 60% of saturation(w/v), After centrifugation and resolubilization in NET buffer, capsidprotein was loaded on a Sepharose CL-6B column for rechromatography.

Dialysis and Drying

The peak fractions obtained above were pooled and extensively dialysedagainst sterile water, and lyophilized for storage.

Expression and Purification Qβ-240

Cells (E. coli JM 109, transformed with the plasmid pQβ-240) wereresuspended in LB, sonicated five times for 15 seconds (water icejacket) and centrifuged at 13000 rpm for one hour. The supernatant wasstored at 4° C. until further processing, while the debris were washed 2times with 9 ml of LB, and finally with 9 ml of 0.7 M urea in LB. Allsupernatants were pooled, and loaded on the Sepharose CL-4B column. Thepooled peak fractions were precipitated with ammonium sulphate andcentrifuged. The resolubilized protein was then purified further on aSepharose 28 column and finally on a Sepharose 6B column. The capsidpeak was finally extensively dialyzed against water and lyophilized asdescribed above. The assembly of the coat protein into a capsid wasconfirmed by electron microscopy.

Expression and Purification Qβ-243

Cells (E. coli RR1) were resuspended in LB and processed as described inthe general procedure. The protein was purified by two successive gelfiltration steps on the sepharose CL-4B column and finally on asepharose CL-2B column. Peak fractions were pooled and lyophilized asdescribed above. The assembly of the coat protein into a capsid wasconfirmed by electron microscopy.

Expression and Purification of Qβ-250

Cells (E. coli JM 109, transformed with pQβ-250) were resuspended in LBand processed as described above. The protein was purified by gelfiltration on a Sepharose CL-4B and finally on a Sepharose CL-2B column,and lyophilized as described above. The assembly of the coat proteininto a capsid was confirmed by electron microscopy.

Expression and Purification of Qβ-259

Cells (E. coli JM 109, transformed with pQβ-259) were resuspended in LBand sonicated. The debris were washed once with 10 ml of LB and a secondtime with 10 ml of 0.7 M urea in LB. The protein was purified by twogel-filtration chromatography steps, on a Sepharose CL-4 B column. Theprotein was dialyzed and lyophilized, as described above. The assemblyof the coat protein into a capsid was confirmed by electron microscopy.

B. Cloning, Expression and Purification of Recombinant AP205 VLP Cloningof the AP205 Coat Protein Gene

The cDNA of AP205 coat protein (CP) (SEQ ID NO: 28) was assembled fromtwo cDNA fragments generated from phage AP205 RNA by using a reversetranscription-PCR technique and cloning in the commercial plasmid pCR4-TOPO for sequencing. Reverse transcription techniques are well knownto those of ordinary skill in the relevant art. The first fragment,contained in plasmid p205-246, contained 269 nucleotides upstream of theCP sequence and 74 nucleotides coding for the first 24 N-terminal aminoacids of the CP. The second fragment, contained in plasmid p205-262,contained 364 nucleotides coding for amino acids 12-131 of CP and anadditional 162 nucleotides downstream of the CP sequence. Both p205-246and p205-262 were a generous gift from J. Klovins.

The plasmid 283-58 was designed by two-step PCR, in order to fuse bothCP fragments from plasmids p205-246 and p205-262 in one full-length CPsequence.

An upstream primer p1.44 containing the NcoI site for cloning intoplasmid pQb185, or p1.45 containing the XbaI site for cloning intoplasmid pQb10, and a downstream primer p1.46 containing the HindIIIrestriction site were used (recognition sequence of the restrictionenzyme underlined):

p1.44 (SEQ ID NO: 79) 5′-AACC ATG GCA AAT AAG CCA ATG CAA CCG-3′ p1.45(SEQ ID NO: 80) 5′-AATCTAGAATTTTCTGCGCACCCATCCCGG-3′ p1.46(SEQ ID NO: 81) 5′-AAAAGC TTA AGC AGT AGT ATC AGA CGA TAC G-3′

Two additional primers, p1.47, annealing at the 5′ end of the fragmentcontained in p205-262, and p1.48, annealing at the 3′ end of thefragment contained in plasmid p205-246 were used to amplify thefragments in the first PCR. Primers p1.47 and p1.48 are complementary toeach other.

p1.47: (SEQ ID NO: 82) 5′-GAGTGATCCAACTCGTTTATCAACTACATTT- TCAGCAAGTCTG-3′ p1.48  (SEQ ID NO: 83)5′-CAGACTTGCTGAAAATGTAGTTGATAAACGA- GTTGGATCACTC-3′

In the first two PCR reactions, two fragments were generated. The firstfragment was generated with primers p1.45 and p1.48 and templatep205-246. The second fragment was generated with primers p1.47 andp1.46, and template p205-262. Both fragments were used as templates forthe second PCR reaction, a splice-overlap extension, with the primercombination p1.45 and p1.46 or p1.44 and p1.46. The product of the twosecond-step PCR reactions were digested with XbaI or NcoI respectively,and HindIII, and cloned with the same restriction sites into pQb10 orpQb185 respectively, two pGEM-derived expression vectors under thecontrol of E. coli tryptophan operon promoter.

Two plasmids were obtained, pAP283-58 (SEQ ID NO: 27), containing thegene coding for wt AP205 CP (SEQ ID NO: 28) in pQb10, and pAP281-32 (SEQID NO: 30) with mutation Pro5→Thr (SEQ ID NO: 29), in pQb185. The coatprotein sequences were verified by DNA sequencing. PAP283-58 contains 49nucleotides upstream of the ATG codon of the CP, downstream of the XbaIsite, and contains the putative original ribosomal binding site of thecoat protein mRNA.

Expression and Purification of Recombinant AP205 VLP A. Expression ofRecombinant AP205 VLP

E. coli JM109 was transformed with plasmid pAP283-58. 5 ml of LB liquidmedium with 20 μg/ml ampicillin were inoculated with a single colony,and incubated at 37° C. for 16-24 h without shaking.

The prepared inoculum was diluted 1:100 in 100-300 ml of LB medium,containing 20 μg/ml ampicillin and incubated at 37° C. overnight withoutshaking. The resulting second inoculum was diluted 1:50 in 2TY medium,containing 0.2% glucose and phosphate for buffering, and incubated at37° C. overnight on a shaker. Cells were harvested by centrifugation andfrozen at −80° C.

B. Purification of Recombinant AP205 VLP

Solutions and Buffers:

1. Lysis buffer

-   -   50 mM Tris-HCl pH 8.0 with 5 mM EDTA, 0.1% tritonX100 and PMSF        at 5 micrograms per ml.

2. SAS

-   -   Saturated ammonium sulphate in water

3. Buffer NE.

-   -   20 mM Tris-HCl, pH 7.8 with 5 mM EDTA and 150 mM NaCl.

4. PEG

-   -   40% (w/v) polyethylenglycol 6000 in NET

Lysis:

Frozen cells were resuspended in lysis buffer at 2 ml/g cells. Themixture was sonicated with 22 kH five times for 15 seconds, withintervals of 1 min to cool the solution on ice. The lysate was thencentrifuged for 20 minutes at 12 000 rpm, using a F34-6-38 rotor(Ependorf). The centrifugation steps described below were all performedusing the same rotor, except otherwise stated. The supernatant wasstored at 4° C., while cell debris were washed twice with lysis buffer.After centrifugation, the supernatants of the lysate and wash fractionswere pooled.

Ammonium-sulphate precipitation can be further used to purify AP205 VLP.In a first step, a concentration of ammonium-sulphate at which AP205 VLPdoes not precipitate is chosen. The resulting pellet is discarded. Inthe next step, an ammonium sulphate concentration at which AP205 VLPquantitatively precipitates is selected, and AP205 VLP is isolated fromthe pellet of this precipitation step by centrifugation (14 000 rpm, for20 min). The obtained pellet is solubilised in NET buffer.

Chromatography:

The capsid protein from the pooled supernatants was loaded on aSepharose 4B column (2.8×70 cm), and eluted with NET buffer, at 4ml/hour/fraction. Fractions 28-40 were collected, and precipitated withammonium sulphate at 60% saturation. The fractions were analyzed bySDS-PAGE and Western Blot with an antiserum specific for AP205 prior toprecipitation. The pellet isolated by centrifugation was resolubilizedin NET buffer, and loaded on a Sepharose 2B column (2.3×65 cm), elutedat 3 ml/h/fraction. Fractions were analysed by SDS-PAGE, and fractions44-50 were collected, pooled and precipitated with ammonium sulphate at60% saturation. The pellet isolated by centrifugation was resolubilizedin NET buffer, and purified on a Sepharose 68 column (2.5×47 cm), elutedat 3 ml/hour/fraction. The fractions were analysed by SDS-PAGE.Fractions 23-27 were collected, the salt concentration adjusted to 0.5M, and precipitated with PEG 6000, added from a 40% stock in water andto a final concentration of 13.3%. The pellet isolated by centrifugationwas resolubilized in NET buffer, and loaded on the same Sepharose 28column as above, eluted in the same manner. Fractions 43-53 werecollected, and precipitated with ammonium sulphate at a saturation of60%. The pellet isolated by centrifugation was resolubilized in water,and the obtained protein solution was extensively dialyzed againstwater. About 10 mg of purified protein per gram of cells could beisolated.

Examination of the virus-like particles in Electron microscopy showedthat they were identical to the phage particles.

Example 2 Insertion of a Peptide Containing a Lysine Residue into thec/e1 Epitope of HBcAg(1-149)

The c/e1 epitope (residues 72 to 88) of HBcAg is located in the tipregion on the surface of the Hepatitis B virus capsid (HBcAg). A part ofthis region (Proline 79 and Alanine 80) was genetically replaced by thepeptide Gly-Gly-Lys-Gly-Gly (SEQ ID NO: 33), resulting in the HBcAg-Lysconstruct (SEQ ID NO: 26). The introduced Lysine residue contains areactive amino group in its side chain that can be used forintermolecular chemical crosslinking of HBcAg particles with any antigencontaining a free cysteine group.

HBcAg-Lys DNA, having the amino acid sequence shown in SEQ ID NO:78, wasgenerated by PCRs: The two fragments encoding HBcAg fragments (aminoacid residues 1 to 78 and 81 to 149) were amplified separately by PCR.The primers used for these PCRs also introduced a DNA sequence encodingthe Gly-Gly-Lys-Gly-Gly peptide (SEQ ID NO: 33). The HBcAg (1 to 78)fragment was amplified from pEco63 using primers EcoRIHBcAg(s) andLys-HBcAg(as). The HBcAg (81 to 149) fragment was amplified from pEco63using primers Lys-HBcAg(s) and HBcAg(1-149)Hind(as). PrimersLys-HBcAg(as) and Lys-HBcAg(s) introduced complementary DNA sequences atthe ends of the two PCR products allowing fusion of the two PCR productsin a subsequent assembly PCR. The assembled fragments were amplified byPCR using primers EcoRIHBcAg(s) and HbcAg(1-149)Hind(as).

For the PCRs, 100 pmol of each oligo and 50 ng of the template DNAs wereused in the 50 ml reaction mixtures with 2 units of Pwo polymerase. 0.1mM dNTPs and 2 mM MgSO₄. For both reactions, temperature cycling wascarried out as follows: 94° C. for 2 minutes; 30 cycles of 94° C. (1minute), 50° C. (1 minute), 72° C. (2 minutes).

Primer sequences: EcoRIHBcAg(s): (5′-CCGGAATTCATGGACATTGACCCTTATAAAG-3′) (SEQ ID NO: 58); Lys-HBcAg(as):(5′CCTAGAGCCACCTTTGCCACCATCTTCTAAATT AGTACCCACCCAGGTAGC-3′(SEQ ID NO: 59);                 Lys-HBcAg(s):(5′-GAAGATGGTGGCAAAGGTGGCTCTAGGGACC-TAGTAGTCAGTTATGTC-3′) (SEQ ID NO: 60);                HBcAg(1-149)Hind(as): (5′-CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGAAG-3′) (SEQ ID NO: 61).                         

For fusion of the two PCR fragments by PCR 100 pmol of primersEcoRIHBcAg(s) and HBcAg(1-149)Hind(as) were used with 100 ng of the twopurified PCR fragments in a 50 ml reaction mixture containing 2 units ofPwo polymerase, 0.1 mM dNTPs and 2 mM MgSO₄. PCR cycling conditionswere: 94° C. for 2 minutes; 30 cycles of 94° C. (1 minute). 50° C. (1minute), 72° C. (2 minutes). The assembled PCR product was analyzed byagarose gel electrophoresis, purified and digested for 19 hours in anappropriate buffer with EcoRI and HindIII restriction enzymes. Thedigested DNA fragment was ligated into EcoRI/HindIII-digested pKK vectorto generate pKK-HBcAg-Lys expression vector. Insertion of the PCRproduct into the vector was analyzed by EcoRI/HindIII restrictionanalysis and DNA sequencing of the insert.

Example 3 Expression and Purification of HBcAg-Lys

E. coli strains K802 or JM109 were transformed with pKK-HBcAg-Lys. 1 mlof an overnight culture of bacteria was used to innoculate 100 ml of LBmedium containing 100 μg/ml ampicillin. This culture was grown for 4hours at 37° C. until an OD at 600 nm of approximately 0.8 was reached.Induction of the synthesis of HBcAg-Lys was performed by addition ofIPTG to a final concentration of 1 mM. After induction, bacteria werefurther shaken at 37° C. for 4 hours. Bacteria were harvested bycentrifugation at 5000×g for 15 minutes. The pellet was frozen at −80°C. The pellet was thawed and resuspended in bacteria lysis buffer (10 mMNa₂HPO₄, pH 7.0. 30 mM NaCl, 0.25% Tween-20, 10 mM EDTA) supplementedwith 200 μg/ml lysozyme and 10 μl of Benzonase (Merck). Cells wereincubated for 30 minutes at room temperature and disrupted bysonication. E. coli cells harboring pKK-HBcAg-Lys expression plasmid ora control plasmid were used for induction of HBcAg-Lys expression withIPTG. Prior to the addition of IPTG, a sample was removed from thebacteria culture carrying the pKK-HBcAg-Lys plasmid and from a culturecarrying the control plasmid. Four hours after addition of IPTG, sampleswere again removed from the culture containing pKK-HBcAg-Lys and fromthe control culture. Protein expression was monitored by SDS-PAGEfollowed by Coomassie staining.

The lysate was then centrifuged for 30 minutes at 12,000×g in order toremove insoluble cell debris. The supernatant and the pellet wereanalyzed by Western blotting using a monoclonal antibody against HBcAg(YVS1841, purchased from Accurate Chemical and Scientific Corp.,Westbury, N.Y., USA), indicating that a significant amount of HBcAg-Lysprotein was soluble. Briefly, lysates from E. coli cells expressingHBcAg-Lys and from control cells were centrifuged at 14,000×g for 30minutes. Supernatant (=soluble fraction) and pellet (=insolublefraction) were separated and diluted with SDS sample buffer to equalvolumes. Samples were analyzed by SDS-PAGE followed by Western blottingwith anti-HBcAg monoclonal antibody YVS 1841.

The cleared cell lysate was used for step-gradient centrifugation usinga sucrose step gradient consisting of a 4 ml 65% sucrose solutionoverlaid with 3 ml 15% sucrose solution followed by 4 ml of bacteriallysate. The sample was centrifuged for 3 hrs with 100,000×g at 4° C.After centrifugation, 1 ml fractions from the top of the gradient werecollected and analyzed by SDS-PAGE followed by Coomassie staining. TheHBcAg-Lys protein was detected by Coomassie staining.

The HBcAg-Lys protein was enriched at the interface between 15 and 65%sucrose indicating that it had formed a capsid particle. Most of thebacterial proteins remained in the sucrose-free upper layer of thegradient, therefore step-gradient centrifugation of the HBcAg-Lysparticles led both to enrichment and to a partial purification of theparticles.

Expression and purification of HBcAg-Lys in large scale was performed asfollows. An overnight culture was prepared by inoculating a singlecolony in 100 ml LB, 100 μg/ml Ampicillin and growing the cultureovernight at 37° C. 25 ml of the preculture were diluted in 800 ml LBAmpicillin medium the next day, and the culture grown to an opticaldensity OD⁶⁰⁰ of 0.6-0.8. The culture was then induced with 1 mM IPTG,and left to grow for another 4 hours. The cells were harvested and lysedessentially as described above,

HBcAg-Lys was then purified by first precipitating the protein withammonium sulphate (30% saturation) from the cleared cell lysate, thenloading the resolubilized pellet on a gel filtration column (SephacrylS-400, Pharmacia). The pooled fractions were precipitated again withammonium sulphate, the pellet resolubilized and loaded a second time onthe same gel filtration column. The fractions were finally pooled andconcentrated, and the concentration assessed using a Bradford test(BioRad).

Example 4 Construction of a HBcAg Devoid of Free Cysteine Residues andContaining an Inserted Lysine Residue

A Hepatitis core Antigen (HBcAg), referred to herein asHBcAg-lys-2cys-Mut, devoid of cysteine residues at positionscorresponding to 48 and 107 in SEQ ID NO:25 and containing an insertedlysine residue was constructed using the following methods.

The two mutations were introduced by first separately amplifying threefragments of the HBcAg-Lys gene prepared as described above in Example 2with the following PCR primer combinations. PCR methods and conventionalcloning techniques were used to prepare the HBcAg-lys-2cys-Mut gene.

In brief, the following primers were used to prepare fragment 1:

Primer 1: EcoRIHBcAg(s) (SEQ ID NO: 58) CCGGAATTCATGGACATTGACCCTTATAAAG Primer 2: 48as (SEQ ID NO: 62) GTGCAGTATGGTGAGGTGAGGAATGCTCAGGAGACTC

The following primers were used to prepare fragment 2:

Primer 3: 48s (SEQ ID NO: 63) GSGTCTCCTGAGCATTCCTCACCTCACCATACTGCACPrimer 4: 107as (SEQ ID NO: 64) CTTCCAAAAGTGAGGGAAGAAATGTGAAACCAC

The following primers were used to prepare fragment 3:

Primer 5: HBcAg149hind-as (SEQ ID NO: 65)CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGAA GCGTTGATAG Primer 6: 107s(SEQ ID NO: 66) GTGGTTTCACATTTCTTCCCTCACTTTTGGAAG

Fragments 1 and 2 were then combined with PCR primers EcoRIHBcAg(s) and107 as to give fragment 4. Fragment 4 and fragment 3 were then combinedwith primers EcoRIHBcAg(s) and HBcAg149hind-as to produce the fulllength gene. The full length gene was then digested with the EcoRL(GAATTC) and HindIII (AAGCTT) enzymes and cloned into the pKK vector(Pharmacia) cut at the same restriction sites. Expression andpurification of HBcAg-lys-2cys-Mut were performed as set out in Example3.

Example 5 Construction of HBcAg1-185-Lys

Hepatitis core Antigen (HBcAg) 1-185 was modified as described inExample 2. A part of the c/e1 epitope (residues 72 to 88) region(Proline 79 and Alanine 80) was genetically replaced by the peptideGly-Gly-Lys-Gly-Gly (SEQ ID NO: 33), resulting in the HBcAg-Lysconstruct (SEQ ID NO: 26). The introduced Lysine residue contains areactive amino group in its side chain that can be used forintermolecular chemical crosslinking of HBcAg particles with any antigencontaining a free cysteine group. PCR methods and conventional cloningtechniques were used to prepare the HBcAg1-185-Lys gene.

The Gly-Gly-Lys-Gly-Gly sequence (SEQ ID NO: 33) was inserted byamplifying two separate fragments of the HBcAg gene from pEco63, asdescribed above in Example 2 and subsequently fusing the two fragmentsby PCR to assemble the full length gene. The following PCR primercombinations were used:

fragment I:

Primer 1: EcoRIHBcAg(s) (SEQ ID NO: 58) (see Example 2) Primer 2:Lys-HBcAg(as) (SEQ ID NO: 59) (see Example 2)

fragment 2:

Primer 3: Lys-HBcAg(s) (SEQ ID NO: 60) (see Example 2) Primer 4:HBcAgwtHindIII CCGTCCCAAGCTTCTAACATTGAGATTCCCGAGATTG (SEQ ID NO: 67)Assembly:

Primer 1: EcoRIHBcAg(s) (SEQ ID NO: 58) (see example 2)

Primer 2: HBcAgwtHindIII (SEQ ID NO: 67)

The assembled full length gene was then digested with the EcoRI (GAATTC)and HindIII (AAGCTT) enzymes and cloned into the pKK vector (Pharmacia)cut at the same restriction sites.

Example 6 Fusion of a Peptide Epitope in the MIR Region of HbcAg

The residues 79 and 80 of HBcAg1-185 were substituted with the epitopeCεH3 of sequence VNLTWSRASG (SEQ ID NO: 68). The CεH3 sequence stemsfrom the sequence of the third constant domain of the heavy chain ofhuman IgE. The epitope was inserted in the HBcAg1-185 sequence using anassembly PCR method. In the first PCR step, the HBcAg1-185 geneoriginating from ATCC clone pEco63 and amplified with primers HBcAg-wtEcoRI fwd and HBcAg-wt Hind III rev was used as template in two separatereactions to amplify two fragments containing sequence elements codingfor the CεH3 sequence. These two fragments were then assembled in asecond PCR step, in an assembly PCR reaction.

Primer combinations in the first PCR step: CεH3fwd with HBcAg-wt HindIII rev, and HBcAg-wt EcoRI fwd with CεH3rev. In the assembly PCRreaction, the two fragments isolated in the first PCR step were firstassembled during 3 PCR cycles without outer primers, which were addedafterwards to the reaction mixture for the next 25 cycles. Outerprimers: HBcAg-wt EcoRI fwd and HBcAg-wt Hind III rev.

The PCR product was cloned in the pKK223.3 using the EcoRI and HindIIIsites, for expression in E. coli (see Example 2). The chimeric VLP wasexpressed in E. coli and purified as described in Example 2. The elutionvolume at which the HBcAg1-185-CεH3 eluted from the gel filtrationshowed assembly of the fusion proteins to a chimeric VLP.

Primer sequences: CεH3fwd: (SEQ ID NO: 69)5′GTT AAC TTG ACC TGG TCT CGT GCT TCT GGT   GCA TCC AGG GAT CTA GTA GTC 3′ (SEQ ID NO: 70)V N L T W S R A S G A80 S R D L V V86 CεH3rev: (SEQ ID NO: 71)5′ACC AGA AGC ACG AGA CCA GGT CAA GTT AAC  ATC TTC CAA ATT ATT ACC CAC 3′ (SEQ ID NO: 72)                        D78 E L N N G V72 HBcAg-wt EcoRI fwd:(SEQ ID NO: 73) 5′CCGgaattcATGGACATTGACCCTTATAAAG HBcAg-wt Hind III rev:(SEQ ID NO: 74) 5′CGCGTCCCaagcttCTAACATTGAGATTCCCGAGATTG

Example 7 Fusion of Aβ1-6 Peptide in the MIR Region of HbcAg

The residues 79 and 80 of HBcAg1-185 are substituted with the Aβ1-6peptide of sequence: DAEFRH (SEQ ID NO: 75) or DAEFGH (SEQ ID NO: 76).Two overlapping primers are designed using the same strategy describedin Example 6, and the fusion protein constructed by assembly PCR. ThePCR product is cloned in the pKK223.3 vector, and expressed in E. coliK802. The chimeric VLPs are expressed and purified as described inExample 3.

Example 8 Fusion of a Aβ1-6 Peptide to the C-Terminus of the Qβ A1Protein Truncated at Position 19 of the CP Extension

A primer annealing to the 5′ end of the Qβ A1 gene and a primerannealing to the 3′ end of the A1 gene and comprising additionally asequence element coding for the Aβ1-6 peptide, of sequence DAEFRH (SEQID NO: 75) or DAEFGH (SEQ ID NO: 76), are used in a PCR reaction withpQβ10 as template. The PCR product is cloned in pQβ10 (Kozlovska T. M.et al., Gene 137: 133-37 (1993)), and the chimeric VLP expressed andpurified as described in Example 1.

Example 9 Insertion of a Aβ1-6 Peptide Between Positions 2 and 3 of frCoat Protein

Complementary primers coding for the sequence of the Aβ1-6 peptide ofsequence DAEFRH (SEQ ID NO: 75) or DAEFGH (SEQ ID NO: 76), andcontaining Bsp119I compatible ends and additional nucleotides enablingin frame insertion, are inserted in the Bsp 119I site of the pFrd8vector (Pushko, P. et al., Prot. Eng. 6: 883-91 (1993)) by standardmolecular biology techniques. Alternatively, the overhangs of the pFrd8vector are filled in with Klenow after digestion with Bsp119I, andoligonucleotides coding for the sequence of the Aβ1-6 peptide andadditional nucleotides for in frame cloning are ligated in pFrd8 afterthe Klenow treatment. Clones with the insert in the right orientationare analysed by sequencing. Expression and purification of the chimericfusion protein in E. coli JM109 or E. coli K802 is performed asdescribed in Pushko, P. et al, Prot. Eng. 6:883-91 (1993), but for thechromatography steps which are performed using a Sepharose CL-4B orSephacryl S-400 (Pharmacia) column. The cell lysate is precipitated withammonium sulphate, and purified by two successive gel filtrationpurification steps, similarly to the procedure described for Qβ inExample 1.

Example 10 Insertion of a Aβ1-6 Peptide Between Positions 67 and 68 ofTy1 Protein p1 in the Vector pOGS8111

Two complementary oligonucleotides coding for the Aβ1-6 peptide, ofsequence DAEFRH (SEQ ID NO: 75) or DAEFGH (SEQ ID NO: 76), with endscompatible with the NheI site of pOGS8111 are synthesized. Additionalnucleotides are added to allow for in frame insertion of a sequencecoding for the Aβ1-6 peptide according to the description of EP 677,111.The amino acids AS and SS flanking the inserted epitope are encoded bythe altered NheI sites resulting from the insertion of theoligonucleotide in the TyA(d) gene of pOGS8111.

POGS8111 is transformed into S. cervisiae strain MC2, for expression ofthe chimeric Ty VLP as described in EP0677111 and references therein.The chimeric Ty VLP is purified by sucrose gradient ultracentrifugationas described in EP 677,111.

Example 11 Insertion of a Aβ1-6 Peptide into the Major Capsid Protein L1of Papillomavirus Type 1 (BPV-1)

A sequence coding for the Aβ1-6 peptide having the sequence DAEFRH (SEQID NO: 75) or DAEFGH (SEQ ID NO: 76) is substituted to the sequencecoding for amino acids 130-136 of the BPV-1 L1 gene cloned in thepFastBac1 (GIBCO/BRL) vector as described (Chackerian, B. et al., Proc.Natl. Acad. USA 96: 2373-2378 (1999)). The sequence of the construct isverified by nucleotide sequence analysis. Recombinant baculovirus isgenerated using the GIBCO/BRL baculovirus system as described by themanufacturer. The chimeric VLPs are purified from baculovirus infectedSf9 cells as described by Kirnbauer, R. et al., Proc. Natl. Acad. Sci.89:12180-84 (1992) and Greenstone, H. L., et al., Proc. Natl. Acad. Sci.95:1800-05 (1998).

Example 12 Immunization of Mice with Aβ1-6 Peptide Fused to VLPs

Chimeric VLPs displaying the Aβ1-6 peptide of sequence DAEFRH (SEQ IDNO: 75) or DAEFGH (SEQ ID NO: 76) generated in Examples 7-11 are usedfor immunization of human transgenic APP mice or C57/BL6 mice asdescribed in Example 13 and 14. The sera obtained from the immunizedmice are analysed in a Aβ1-6 peptide or Aβ1-40 or Aβ1-42 specific ELISAas described in Example 13.

The protective effect of the vaccine is examined by immunizing a largegroup of human APP transgenic mice as described in Example 14.

Example 13 Coupling of Aβ1-6 Peptide to Qβ VLP (QβAβ1-6), andImmunization of Mice with QβAβ1-6 A. Coupling of Aβ1-6 Peptide Qβ VLP

The Aβ1-6 peptide (sequence: NH2-DAEFRHGGC-CONH2) (SEQ ID NO: 77) waschemically synthesized; the initial NH2 group indicates that the peptidehas a free N-terminus, and the terminal NH2 group indicates that thepeptide has an amidated carboxy-terminus. Qβ VLP was expressed andpurified as described in example 1. Qβ VLP, in 20 mM Hepes, 150 mM NaCl.pH 8.2 (HBS, pH 8.2) was reacted at a concentration of 2 mg/ml(determined in a Bradford assay), with 1.43 mM SMPH (Pierce, RockfordIll.), diluted from a stock in DMSO, for 30 minutes at room temperature(RT). The reaction mixture was then dialyzed against HBS, pH 8.2 bufferat 4° C., and reacted with 0.36 mM of Aβ1-6 peptide, diluted in thereaction mixture from a 50 mM stock in DMSO. The coupling reaction wasleft to proceed for 2 hours at 15° C., and the reaction mixture dialyzed2×2 hours against a 1000-fold volume HBS, pH 8.2, and flash frozen inliquid nitrogen in aliquots for storage at −80° C. until further use.

An aliquot was thawed, and coupling of the Aβ1-6 peptide to the Qβ VLPsubunits assessed by SDS-PAGE and the protein concentration measured ina Bradford assay. The result of the coupling reactions are shown in FIG.1.

FIG. 1 shows the SDS-PAGE analysis of the coupling reaction of Aβ1-6peptide and Qβ VLP. The samples were run under reducing conditions on a16% Tris-glycine gel, stained with coomassie brilliant blue. Lane 1 isthe protein marker, with corresponding molecular weights indicated onthe left border of the gel; lane 2, derivatized Qβ VLP protein; lane 3,the supernatant of the coupling reaction of Qβ VLP protein to the Aβ1-6peptide; lane 4, the pellet of the coupling reaction of Qβ VLP proteinto the Aβ1-6 peptide; Coupling products corresponding to the coupling of1, 2 and 3 peptides per monomer are indicated by arrows in the Figure.More than 1.5 peptides per subunit were coupled on average; nearly nosubunits were left uncoupled.

B. Immunisation of Mice with Aβ1-6 Peptide Coupled to Qβ VLP andAnalysis of Immune Response

Qβ VLP coupled to Aβ1-6 peptide (denominated here Qb-Ab-1-6) wasinjected s.c. in mice (3 mice) at day 0 and 14. Aβ1-6 peptide wascoupled to Qβ VLP protein as described above. Each mice (C57BL6) wasimmunized with 10 μg of vaccine diluted in PBS to 200 μl. Mice wereretroorbitally bled on day 21, and the titer of the antibodies specificfor the Aβ1-6 peptide were measured in an ELISA against Aβ1-6. The Aβ1-6peptide was coupled to bovine RNAse A using the chemical cross-linkersulfo-SPDP. ELISA plates were coated with coupled RNAse preparations ata concentration of 10 μg/ml. The plates were blocked and then incubatedwith serially diluted mouse sera. Bound antibodies were detected withenzymatically labeled anti-mouse IgG antibodies. As a control, preimmunesera of the same mice were also tested. The results are shown in FIG. 2.

FIG. 2 shows an ELISA analysis of the IgG antibodies specific for Aβ1-6peptide in sera of mice immunized against the Aβ1-6 peptide coupled toQβ VLP. The results are shown for the three mice immunized (A1-A3), thepre-immune serum is indicated as “pre” in the figure; the result for onepre-immune serum is shown. Comparison of the pre-immune sera with thesera of mice immunized with “Qb-Ab-1-6” shows that a strong specificantibody response against peptide Aβ1-6 could be obtained in the absenceof adjuvant.

C. ELISA Against Aβ 1-40 Peptide

Human Aβ1-40 or Aβ1-42 peptide stock was made in DMSO and diluted incoating buffer before use. ELISA plates were coated with 0.1 g/well Aβ1-40 peptide. The plates were blocked and then incubated with seriallydiluted mouse serum obtained above. Bound antibodies were detected withenzymatically labeled anti-mouse IgG antibody. As a control, seraobtained before vaccination were also included. The serum dilutionshowing a mean three standard deviations above baseline was calculatedand defined as “ELISA titer”. No specific antibodies were detected inpreimmune sera. The titer obtained for the three mice was of 1:100000,showing a strong specific immune response against Aβ 1-40. Thus,immunization with Aβ1-6 coupled to Qβ VLP elicits strong antibody titerscross-reactive with Aβ1-40.

FIG. 3 shows the result of the ELISA. The ELISA signal as the opticaldensity at 405 nm, obtained for the sera of three mice (A1-A3) immunizedwith Aβ1-6 peptide coupled to Qβ VLP as described above, is plotted foreach of the dilutions, indicated on the x-axis. The result for the threemice bled at day 21 is shown. Also included is a pre-immune serum. Thetiter of the antibodies in the sera was determined as described above,and was of 1:100000 for all three mice.

Example 14 Immunization of Human APP Transgenic Mice

8 months old female APP23 mice which carry a human APP transgene(Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA 94: 13287-13292(1997)) are used for vaccination. The mice are injected subcutaneouslywith 25 μg vaccine diluted in sterile PBS and 14 days later boosted withthe same amount of vaccine. Mice are bled from the tail vein before thestart of immunization and 7 days after the booster injection. The seraare analyzed for the presence of antibodies specific to a Aβ1-6, toAβ1-40 and Aβ1-42 by ELISA as described in Example 13.

Example 15 Coupling of Murine Aβ1-6 to Qβ VLP, Injection of the Vaccinein Mice, and Analysis of the Immune Response

Murine Aβ1-6 peptide (sequence: NH2-DAEFGHGGC-CONH2) (SEQ ID NO: 78) ischemically synthesized, and used for coupling to Qβ VLP as described inExample 13. The vaccine is injected in C57BL/6 mice, and the titer ofthe elicited antibodies against murine Aβ1-6, murine Aβ1-40 and murineAβ1-42 determined. The immunization and the ELISA determination areperformed as described in Example 13.

Example 16 Binding of Sera Elicited Against Aβ1-6 to Human APPTransgenic Mice Plaques and AD Plaques Immunohistochemistry in BrainSlices

Consecutive paraffin brain sections of a 18 months, old heterozygousAPP23 mouse and entorhinal cortex sections from an AD patient BraakStage III (Institute of Pathology, University Basel) were used forstaining. Antigenicity was enhanced by treating human brain sectionswith concentrated formic acid for five minutes and mouse brain sectionsby microwave heating at 90° C. for 3 minutes. Mice sera elicited againsthuman A1-6 (obtained as described in Example 13) were diluted 1:1000 inPBS with 3% goat serum and incubated over night. Following rinsing,sections were incubated for 1 hour with biotinylated anti mousesecondary antibody diluted 1:200 in PBS. After rinsing, sections werefurther processed with the avidin-biotin-peroxidase technique (ABC-EliteKit PK6100; Vector Laboratories). Finally, sections were reacted withDiaminobenzidine (DAB) metal enhanced substrate (Boehringer, Code1718096), counterstaind with Hemalum, dehydrated, cleared in Xylene andcoversliped.

The result of the histologic stains are shown in FIGS. 4 A and B.Sections were stained with the sera of the three mice immunized againsthuman Aβ1-6 coupled to Qβ VLP. Each serum stained positively the amyloidplaques from transgenic mice and AD. Results for one of the three seraare shown. Sera elicited against human Aβ1-6 clearly stain amyloidplaques of the transgenic human APP23 mouse, as well as amyloid plaquesfrom AD patients. Pre-immune sera were negative. Extracellular amyloidplaques and isolated blood vessels are stained by the antibodies.

Example 17 Specificity of Sera Elicited Against Human A1-6, Assessed byHistology of Mice Plaques Immunohistochemistry in Brain Slices

Consecutive paraffin brain sections of a 3 months and an 18 months oldheterozygous APP23 mouse overexpressing human APP were stained asdescribed in Example 16 with a representative mouse serum elicitedagainst human Aβ1-6 as described in Example 13, or with a rabbitpolyclonal antibody specific for the last 20 amino acids of murine orhuman APP and which therefore does not recognizes Aβ. The sectionsincubated with the rabbit polyclonal antibody were treated as describedin Example 16, except for the use of a biotinylated anti rabbitsecondary antibody (BA 1000, Vector Laboratories).

The result of the histologic stains are shown on FIGS. 5 A, B, C, D andE. Aβ1-6, marked on the bottom left of the sections indicate that seraelicited against Aβ1-6 have been used for the staining, while “Pab”indicates that the sections have been stained with the polyclonalantibody specific for the last 20-amino acids of murine or human APP,corresponding to positions 676-695 in APP₆₉₅.

Comparison of the staining of sections from 18 months old mice (FIGS. 5A and C) shows that the sera elicited against Aβ1-6 do not cross-reactwith APP expressed in the brain, which is however stained by the controlpolyclonal antibody. FIG. 5 B shows a brain section from a 3 months oldmouse, a timepoint where amyloid deposits are not yet visible, stainedwith the polyclonal antibody specific for APP. FIGS. 5 D and 5E show amagnification of the CA1 pyramidal layer of the hippocampus from FIG.5A, and FIG. 5B, respectively.

Example 18 A. Coupling of Aβ1-6 Peptide to fr Capsid Protein

A solution of 120 μM fr capsid protein in 20 mM Hepes, 150 mM NaCl pH7.2 is reacted for 30 minutes with a 10 fold molar excess of SMPH(Pierce), diluted from a stock solution in DMSO, at 25° C. on a rockingshaker. The reaction solution is subsequently dialyzed twice for 2 hoursagainst 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzed frreaction mixture is then reacted with a a five-fold molar excess ofAβ1-6 peptide (sequence: NH2-DAEFRHGGC-CONH2) (SEQ ID NO: 77) for 2hours at 16° C. on a rocking shaker. Coupling products are analysed bySDS-PAGE.

B. Coupling of Aβ1-6 Peptide to HBcAg-Lys-2Cys-Mut

A solution of 1 ml of 120 μM HBcAg-Lys-2cys-Mut in 20 mM Hepes, 150 mMNaCl pH 7.2 is reacted for 30 minutes with a 10 fold molar excess ofSMPH (Pierce), diluted from a stock solution in DMSO, at 25° C. on arocking shaker. The reaction solution is subsequently dialyzed twice for2 hours against 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. Thedialyzed HBcAg-Lys-2cys-Mut reaction mixture is then reacted with afive-fold molar excess of Aβ1-6 peptide (sequence: NH2-DAEFRHGGC-CONH2)(SEQ ID NO: 77) for 2 hours at 16° C. on a rocking shaker. Couplingproducts are analysed by SDS-PAGE.

C. Coupling of Aβ1-6 Peptide to Pili

A solution of 125 μM Type-1 pili of E. coli in 20 mM Hepes, pH 7.4, isreacted for 60 minutes with a 50-fold molar excess of cross-linker SMPH(Pierce), diluted from a stock solution in DMSO, at RT on a rockingshaker. The reaction mixture is desalted on a PD-10 column(Amersham-Pharmacia Biotech). The protein-containing fractions eluatingfrom the column are pooled, and the desalted derivatized pill protein isreacted with a five-fold molar excess of Aβ1-6 peptide (sequence:NH2-DAEFRHGGC-CONH2) (SEQ ID NO: 77) for 2 hours at 16° C. on a rockingshaker. Coupling products are analysed by SDS-PAGE.

D. Immunization of Mice with Aβ1-6 Peptide Coupled to fr-Capsid Protein,HBcAg-Lys-2cys-Mut or Pili

Aβ1-6 peptide coupled to fr-capsid protein, HBcAg-Lys-2cys-Mut or pilias described above is injected s.c. in mice (3 mice) at day 0 and 14.Each mice (C57BL/6) is immunized with 10 μg of vaccine diluted in PBS to200 μl. Mice are retroorbitally bled on day 21, and the titer of theantibodies specific for the Aβ1-6 peptide or Aβ1-40 or Aβ1-42 aremeasured by ELISA as described in Example 13.

Example 19 Immunisation of Rhesus Monkeys with QβhAβ1-6

In order to test induction of antibodies against human Aβ using a humanAβ1-6 peptide based vaccine in the case where Aβ1-6 is a self antigen,rhesus monkeys were immunized with QβhAβ1-6, as the Aβ sequence isidentical between humans and Rhesus monkeys. QβhAβ1-6 vaccine was madeas described in Example 13. Four Rhesus monkeys, between 10 and 15 yearsof age, were immunized at day 0 with 50 μg of vaccine, and boosted twiceat day 28 and 56 with 25 μg of vaccine. The monkeys were immunizedsubcutaneously in the back. The animals were bled at day 0 (prebleed),42 and 70. 4 ml of blood were collected from the V. cephalicaantebrachii. The titer of antibodies specific for Aβ1-40 were measuredby ELISA essentially as described in Example 13, using a secondaryantibody specific for Monkey IgG.

As humans and rhesus monkeys share the same Aβ sequence, the generationof high titer antibodies in rhesus monkeys specific for Aβ1-40 showsthat immunization with hAβ1-6 coupled to Q breaks tolerance against theself-antigen Aβ. Furthermore, antibodies recognizing full length Aβ aregenerated with the coupled Aβ1-6 fragment in primates.

The results of the ELISA are shown in FIG. 6. Plotted in the diagram arethe titers of Aβ1-40 specific antibodies measured in the sera of the 4monkeys (1-4) immunized with QβhAβ1-6 and the average of the titers ofthe 4 monkeys. The titers are represented as OD50 titers. OD50 is thedilution of the antibodies at which the signal reaches half of itsmaximal value. The maximal value (OD max) was obtained from a referenceserum originating from a monkey immunised with QβhAβ1-27 and recognizingAβ1-40 as well and measured on the same ELISA plate.

Two monkeys (described above) were bled at day 97, 110, 117, 124, 138,143, 152, 159, 166, and received a third boost with 25 μg of vaccine atday 110. Sera were pooled (99 ml) and used for affinity purification ofAβ1-6-specific antibodies. These antibodies were used forimmunohistochemical staining at a concentration of 1.5 μg/ml and abiotinylated secondary anti-monkey antibody was used for detection.Paraffine brain sections of 18 months old heterozygous APP23 mouse andan AD patient—Braak Stage III—were used for staining. Plaque-specificstaining was observed both in APP23 mouse brain sections and in the ADpatient brain sections (FIG. 7).

The result of the histological analysis is shown in FIGS. 7 A and B.Depicted in FIG. 7 A is the staining of human APP transgenic mouseplaques (APP23 strain) with the above described affinity purifiedantiserum specific for Aβ1-6. FIG. 7 B shows the staining of human ADplaques with the same purified antiserum. The purified antiserum wasused at a concentration of 1.5 μg/ml in both cases. Typical plaques areindicated by an arrow on both figures.

Example 20 Coupling of Murine Aβ1-6 to AP205 VLP, Immunisation of Miceand Analysis of Immune Response A. Coupling of Murine Aβ1-6 Peptide toAP205 VLP

The peptide murine Aβ1-6 (mAβ1-6, sequence: NIH2-DAEFGHGGC-CONH2 (SEQ IDNO: 78) was chemically synthesized; the initial NH2 group indicates thatthe peptide has a free N-terminus, and the terminal NH2 group indicatesthat the peptide has an amidated carboxy-terminus). AP205 VLP (expressedand purified as described in Example 1), in 20 mM Hepes, 150 mM NaCl, pH8.0 (HBS, pH 8.0) was reacted at a concentration of 2 mg/ml (determinedin a Bradford assay), with 2.86 mM SMPH (Pierce, Rockford Ill.), dilutedfrom a 100 mM stock in DMSO, for 30 minutes at room temperature (RT).The reaction mixture was then dialyzed twice against a 1000-fold volumeof HBS, pH 7.4, at 4° C. for two hours; the resulting dialyzed andderivatized AP205 VLP was flash frozen in liquid nitrogen and stored at−20° C. overnight. Derivatized AP205 VLP was diluted with one volume of20 mM HBS, pH 7.4, and reacted 2 hours at 15° C. under shaking with 719μM mAβ1-6 peptide diluted in the reaction mixture from a 50 mM stock inDMSO. The coupling reaction was dialyzed twice against a 1000-foldvolume HBS, pH 7.4, for 2 hours and overnight. The dialyzed reactionmixture was flash frozen in liquid nitrogen in aliquots for storage at−80° C. until further use.

An aliquot was thawed, and coupling of the mAβ1-6 peptide to the AP205VLP subunits assessed by SDS-PAGE and the protein concentration measuredin a Bradford assay. The result of the coupling reaction is shown inFIG. 8.

FIG. 8 shows the SDS-PAGE analysis of the coupling reaction of mAβ1-6peptide to AP205 VLP. The samples were run under reducing conditions ona 16% Tris-glycine gel and stained with coomassie brilliant blue. Lane 1is the protein marker, with corresponding molecular weights indicated onthe left border of the gel; lane 2, AP205 VLP protein; lane 3,derivatized AP205 VLP; lane 4, the supernatant of the coupling reactionof AP205 VLP to mAβ1-6 peptide; lane 5, the pellet of the couplingreaction of AP205 VLP to mAβ1-6 peptide. No AP205 VLP subunits leftuncoupled could be detected on the gel, while bands corresponding toseveral peptides per subunits were visible, demonstrating a very highcoupling efficiency. In particular, there is much more than one Aβ1-6peptide per AP205 VLP subunit.

B. Immunisation of Mice with mAβ1-6 Peptide Coupled to AP205 VLP andAnalysis of Immune Response

AP205 VLP coupled to mAβ1-6 peptide was injected s.c. in mice (3 mice)at day 0 and 14. mAβ1-6 peptide was coupled to AP205 VLP as describedabove. Each mice (C57BL/6) was immunized with 25 μg of vaccine dilutedin PBS to 200 μl. Mice were retroorbitally bled on day 21, and the titerof the antibodies specific for the mAβ1-6 peptide were measured in anELISA against mAβ1-6. The mAβ1-6 peptide was coupled to bovine RNAse Ausing the chemical cross-linker sulfo-SPDP. ELISA plates were coatedwith preparations of RNAse-mAβ1-6 at a concentration of 10 μg/ml. Theplates were blocked and then incubated with serially diluted mouse sera.Bound antibodies were detected with enzymatically labeled anti-mouse IgGantibodies. As a control, preimmune sera of the same mice were alsotested. The results are shown in FIG. 9.

FIG. 9 shows an ELISA analysis of the IgG antibodies specific for mAβ1-6peptide in sera of mice immunized with the mAβ1-6 peptide coupled toAP205 VLP. The results are shown for the sera of the three immunizedmice collected at day 21 (A1 d21-A3 d21), the pre-immune serum isindicated as “pre imm” in the figure; the result for one pre-immuneserum is shown. Comparison of the pre-immune serum with the sera of themice immunized with mAβ1-6 coupled to AP205 VLP shows that a strongspecific antibody response against peptide mAβ1-6, which is aself-antigen, could be obtained in the absence of adjuvant. Furthermore,coupling of a self-peptide to AP205 VLP leads to break of toleranceagainst this peptide, and to a very high specific immune response. Thus,AP205 VLP is suitable for generating high antibody titers against Aβpeptides in the absence of adjuvant.

Example 21 Immunisation with QβhAβ1-6 Reduces Amyloid Plaques inTransgenic Mice Over-Expressing the “Swedish/London” Mutant AmyloidPrecursor Protein

This example demonstrates that immunization with QβhAβ1-6 in a mousemodel developing Alzheimer's disease-like diffuse (Congo-Red negative)amyloid plaques, resulted in a massive reduction of plaque density inneocortical and subcortical brain areas. Histological occurrence ofdiffuse amyloid plaques is a prominent feature of AD brain pathology(Selkoe, 1994. Annu. Rev. Neurosci. 17:489-517) and, therefore, theexample demonstrates that immunization with QβhAβ1-6 provides aneffective approach for the treatment of Alzheimer's disease.

To evaluate the therapeutic efficacy of immunization with Qβ-Aβ1-6transgenic mice over-expressing the “Swedish/London” mutant amyloidprecursor protein under the control of the mouse Thy-1 promoter (APP24;K670N/M671L; V717I, patent No. WO0980-36-4423) were used. This mousestrain is characterized by a large number amyloid plaques in theneocortex, hippocampus, caudate putamen, and thalamus at an age of 18months. Plaques can be first observed at an age of 9 months.Histologically, the amyloid plaques in APP24 mice are predominantly of adiffuse type, i.e. they are negative in Congo-Red staining. To a lesserdegree, also compact amyloid plaques (Congo-Red positive) can be found.

Human Aβ1-6 peptide coupled to Qβ VLP (QβhAβ1-6) was made as describedin Example 13. In terms of the experimental procedure followed, which isnot necessary for describing or enabling the invention, APP24 transgenicmice 9.5 months of age were injected subcutaneously at day 0 with 25 μgof QβhAβ1-6 in phosphate-buffered saline (PBS) (administered as 2×100 μlper mouse) (n=16) or as negative controls with PBS (administered as2×100 μl per mouse) (n=9) or with Qβ virus-like particle devoid ofcoupled antigen (n=11). Mice were subsequently injected 25 μg ofQβhAβ1-6-vaccine, Qβ, or PBS on day 15, 49, 76, 106, 140, 169, 200, 230,259, and 291. Animals were bled 1-2 days before the first immunization(day 0) and on day 56, 90, 118, 188, 214, 246, and 272 via the tailvein. Blood serum was also collected on day 305, at which time alsobrains were collected for histopathology (age of the mice at this timepoint: 19.5 months).

The titer of antibodies specific for Aβ1-40 were measured by ELISAessentially as described in Example 13. The results of the ELISA areshown in FIG. 10. Plotted in the diagram are the titers of Aβ1-40 orAβ1-42 specific antibodies measured in the sera of mice immunized withQβhAβ1-6. The titers are represented as OD50% titers. OD50% is thedilution of the antibodies at which the signal reaches half of itsmaximal value. The maximal value (OD max) was obtained from a referenceantibody recognizing Aβ1-40 and Ab42, and measured on the same ELISAplate. All QβhAβ1-6 immunized mice developed OD50% titers above 1:8000(pre-immune serum titers were below 1:100) demonstrating a consistentantibody response to QβhAβ1-6 even in old APP24 mice (FIG. 10). MedianOD50% titers in the immunized group were in the range of 1:20,000 to1:50,000 throughout the immunization period.

For quantification of amyloid plaques, brains were fixed by immersion in4% formaldehyde in 0.1 M PBS at 4° C. After dehydration with ethanol,brains were embedded in paraffin and cut sagitally with a microtome at 4μm thickness. Sections were mounted onto super frost slides and dried at37° C. Sections were washed in PBS and antigenicity enhanced bymicrowave heating at 90° C. for 3 minutes in 0.1 M citric acid buffer.NT11 antisera (anti Aβ1-40, Sturchler-Pierrat et al., 1997, Proc. Natl.Aced. Sci. 94: 13287-13292) were diluted 1:1000 in PBS with 3% goatserum and incubated over night at 4° C. Following rinsing, sections wereincubated for 1 hour with biotinylated anti rabbit IgG secondaryantibody (BA 1000, Vector Laboratories) diluted 1:200 in PBS. Afterrinsing, sections were further processed with theavidin-biotin-peroxidase technique (ABC-Elite Kit PK6100; VectorLaboratories). Finally, sections were reacted with Diaminobenzidine(DAB) metal enhanced substrate (Boehringer, Code 1718096),counterstained with Hemalum, dehydrated, cleared in Xylene and coverslipped. Systematic-random series of brain sections at three differentanatomical planes per animal were used for the analysis. Amyloid plaqueswere quantified using an MCID image analyzer (Imaging Research, BrockUniversity, Ontario-Canada, Program Version MS elite). The microscopicimage was digitized by use of a Xillix black and white CCD TV camera andstored with 640×480 pixel resolution at 256 gray levels. The pixel sizewas calibrated using an object micrometer at 5× magnification (LeicaNeoplan Objective). Using a motor driven microscope stage for exactpositioning of adjacent object fields the entire neocortex and olfactorynucleus of each section was analysed. For each object field theanatomical area was defined by manual outline. For each individualsection the sample area was defined by manual threshold setting (greylevel) between immunopositive amyloid plaques and tissue background.Isolated tissue artifacts were excluded by manual outline. Raw data aremeasured as individual counts (amyloid deposits) and proportional areavalues (immunopositive amyloid/cortex or olfactory nucleus).

Data of each mouse were normalized to number of deposits (plaques) permm² and total plaque area in % of the entire neocortex. QβhAβ1-6immunized mice revealed a dramatic reduction of amyloid deposits in thecortex and subcortical areas as compared to either PBS or Qβ injectedcontrol groups (FIG. 11). Both the median number of deposits and thetotal plaque area were highly significantly reduced between 80-98%compared to the PBS group in the cortex, caudate putamen, hippocampus,and thalamus (p<0.001 vs. PBS-group, Mann-Whitney test; FIG. 12).

In a second study, APP24 transgenic mice 13.5 months of age wereinjected subcutaneously at day 0 with 25 μg of QβhAβ1-6 inphosphate-buffered saline (PBS) (administered as 2×100 d per mouse)(n=15) or as negative controls with PBS (administered as 2×100 μl permouse) (n=15). Mice were subsequently injected 25 μg ofQβhAβ1-6-vaccine, or PBS on day 16, 46, 76, 109, 140, and 170. Animalswere bled 1-2 days before the first immunization (day 0) and on day 31,59, 128, and 154 via the tail vein. Blood serum was also collected onday 184, at which time also brains were collected for histopathology(age of the mice at this time point: 19.5 months). The titer ofantibodies specific for Aβ1-40 were determined and expressed asdescribed above and again all immunized mice were found to respond toQβhAβ1-6 immunization with serum OD50% titers at least above 1:2000 (notshown).

Median OD50% titers were in the range of 1:10,000 to 1:50,000 throughoutthe immunization period. Quantification of amyloid deposits was done asdescribed above. Compared to the experiment where immunization wasinitiated earlier (i.e. at an age of 9.5 months) the reduction of plaquedeposit number (−55%) and area (−32%) was less dramatic in theneocortex, but still very pronounced (FIG. 13) and highly significant(p>0.001 vs. PBS, Mann-Whitney test). In subcortical areas plaquedeposit number and area were reduced by 60-90% in the to QβhAβ1-6immunized group. The more pronounced effect in these areas as comparedto the cortex is probably related to the more protracted time course ofplaque formation in these areas.

Taken together, both experiments demonstrate that QβhAβ1-6 immunizationin transgenic mice over-expressing the “Swedish/London” mutant amyloidprecursor protein dramatically reduces the occurrence of amyloiddeposits in these mice.

FIG. 10: Serum anti Aβ40/42 antibody titers (OD50%) in transgenic miceover-expressing the “Swedish/London” mutant amyloid precursor protein.Mice were immunized with QβhAβ1-6 between 9.5 and 19 months of age.Shown are individual values (black dots) and box plots, where the endsof the boxes define the 25^(th) and 75^(th) percentiles, with a line atthe median and error bars defining the 10^(th) and 90^(th) percentiles(outlyers are shown as dots).

FIG. 11: Immunohistochemical staining of amyloid plaques in sagittalbrain sections. The sagittal brain section of a transgenic mouseover-expressing the “Swedish/London” mutant amyloid precursor proteinimmunized with Qβ (A) or QβhAβ1-6 (B) vaccine is shown in the Figure.

FIG. 12: Quantification of plaque deposition in transgenic miceover-expressing the “Swedish/London” mutant amyloid precursor proteinafter immunization between 9.5 and 19 months of age. (A) Cortical plaquedensity. (B) Cortical plaque area. (C) Plaque density in the caudateputamen. (D) Plaque area in the caudate putamen. (E) Plaque density inthe hippocampus. (F) Plaque area in the hippocampus. (G) Plaque densityin the thalamus. (H) Plaque area in the thalamus. Plaque density isexpressed in plaques/mm², plaque area in percent of tissue area coveredby amyloid beta. Data are shown as individual values (black dots) andbox plot. The ends of the boxes define the 25^(th) and 75^(th)percentiles, with a line at the median and error bars defining the10^(th) and 90^(th) percentiles. ** p<0.001 (Mann Whitney Rank SumTest). PBS, n=9, Qβ, n=11, QβhAβ1-6, n=16.

Example 22 Immunisation with QβhAβ1-6 Reduces Amyloid Plaques inTransgenic Mice Over-Expressing the “Swedish” Mutant Amyloid PrecursorProtein

This example demonstrates that immunization with QβhAβ1-6 provides aneffective approach for the treatment of Alzheimer's disease even whenthe immunization is initiated in a very advanced stage of amyloid plaquepathology. The amyloid plaque deposition process in the AD mouse modelused in this example starts already at an age about 6 months(Sturchler-Pierrat et al., 1997, Proc. Natl. Acad. Sci. 94:13287-13292). In the study described herein, immunization with QβhAβ1-6was initiated at an age of 18 months, where already a high number ofcompact plaques had been formed in the cortex. The example alsodemonstrates the ability of QβhAβ1-6 to induce Aβ40/42 antibodies invery aged animals (no non-responders in 19 immunized mice).

To evaluate the therapeutic effects of immunization with QβhAβ1-6transgenic mice over-expressing the “Swedish” mutant amyloid precursorprotein (APP23; K670N/M671L, Sturchler-Pierrat et al., 1997, Proc. Natl.Acad. Sci. 94: 13287-13292) were used. The Alzheimer's-like pathology inthese mice has been extensively characterized (Calhoun et al., 1998,Nature 395: 755-756; Phinney et al., 1999, J. Neurosci. 19: 8552-8559;Bondolti et al., 2002, J. Neurosci. 22: 515-522).

Human Aβ1-6 peptide coupled to Qβ VLP (QβhAβ1-6) was made as describedin Example 13. In terms of the experimental procedure followed, which isnot necessary for describing or enabling the invention, APP23 transgenicmice 18 months of age were injected subcutaneously at day 0 with 25 μgof QβhAβ1-6 dissolved in phosphate-buffered saline (administered as2×100 μl per mouse) (n=19) or phosphate-buffered saline as a negativecontrol (n=17) and boosted on day 13, 27-34, 61-63, 90-96, and 123-130with 25 μg of vaccine. Animals were bled 1-2 days before the firstimmunization (day 0) and on day 41-45, and day 68 via the tail vein.Blood serum was also collected on day 152-154, at which time also brainswere collected for histopathology (age of the mice at this time point:23 months).

The titer of antibodies specific for Aβ1-40 were measured by ELISAessentially as described in Example 13 and the results expressed asdescribed in Example 21. The results of the ELISA are shown in FIG. 14.All QβhAβ1-6 immunized mice developed OD50% titers above 1:2000(pre-immune serum titers were below 1:100) demonstrating a consistentantibody response to Qβ-Aβ1-6 even in very old mice (FIG. 14). MedianOD50% titers were in the range of 1:9,000 to 1:20,000 throughout theimmunization period.

Quantification of amyloid plaques was done as described in Example 21.Data of each mouse were normalized to number of deposits (plaques) permm² and total plaque area in % of the entire neocortex. QβhAβ1-6immunized mice revealed a smaller number of deposits in the cortex (FIG.15, FIG. 16), mostly due to a reduction of small sized plaques. Comparedto the non-immunized group the median plaque number was reduced by 33%in the QβhAβ1-6 immunized group (p<0.001 vs PBS-group. Mann-Whitneytest). Since mostly small-sized plaques were affected the reduction ofthe total plaque area was moderate and amounted to 10% (p<0.01 vs. PBSgroup, Mann-Whitney test).

FIG. 14: Serum anti Aβ40/42 antibody titers (OD50%) in transgenic miceover-expressing the “Swedish” mutant amyloid precursor protein. Micewere immunized with QβhAβ1-6 between 18 and 23 months of age. Shown areindividual values (black dots) and box plots, where the ends of theboxes define the 25^(th) and 75^(th) percentiles, with a line at themedian and error bars defining the 10^(th) and 90^(th) percentiles(outlyers are shown as dots).

FIG. 15: Immunohistochemical staining of amyloid plaques in sagittalbrain sections. Arrows point to small sized deposits. Shown in theFigure is a sagittal brain section from a transgenic mouseover-expressing the “Swedish” mutant amyloid precursor protein immunizedwith PBS (A) or Q %-Aβ1-6 (B).

FIG. 16: Quantification of plaque deposition in transgenic miceover-expressing the “Swedish” mutant amyloid precursor protein afterimmunization between 18 and 23 months of age. (A) Cortical plaquedensity. (B) Cortical plaque area. Plaque density is expressed inplaques/mm² plaque area in percent of tissue area covered by amyloidbeta, Data are shown as individual values (black dots) and box plot. Theends of the boxes define the 25^(th) and 75^(th) percentiles, with aline at the median and error bars defining the 10^(th) and 90^(th)percentiles. ** p<0.001 (Mann Whitney Rank Sum Test). PBS, n=17,QβhAβ1-6, n=19.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1. An immunogenic composition comprising: (a) a core particle with atleast one first attachment site, wherein said core particle is avirus-like particle of an RNA bacteriophage; and (b) at least oneantigen or antigenic determinant with at least one second attachmentsite, wherein said antigen or antigenic determinant is a Aβ1-6 peptide,and wherein said second attachment site being selected from the groupconsisting of: (i) an attachment site not naturally occurring with saidantigen or antigenic determinant; and (ii) an attachment site naturallyoccurring with said antigen or antigenic determinant, wherein saidsecond attachment site is capable of association to said firstattachment site; and wherein said Aβ1-6 peptide and said core particleinteract through said association to form an ordered and repetitiveantigen array, and further wherein said Aβ1-6 peptide has an amino acidsequence variant of SEQ ID NO:75 (DAEFRH), wherein the amino acid atposition 1 of the SEQ ID NO: 75 variant is aspartic acid, alanine ortyrosine, the amino acid at position 2 of the SEQ ID NO: 75 variant isalanine, serine or tyrosine, the amino acid at position 3 of the SEQ IDNO: 75 variant is glutamic acid, the amino acid at position 4 of the SEQID NO: 75 variant is phenylalanine or tyrosine, the amino acid atposition 5 of the SEQ ID NO: 75 variant is arginine or glycine, and theamino acid at position 6 of the SEQ ID NO: 75 variant is histidine,provided that the full-length sequence of said Aβ1-6 peptide is notDAEFRH.
 2. The immunogenic composition according to claim 1, wherein theamino acid sequence variant of SEQ ID NO: 75 is selected from the groupconsisting of: SEQ ID NO: 76 (DAEFGH), SEQ ID NO: 86 (ASEYRH), and SEQID NO: 90 (YYEFRH).
 3. The immunogenic composition of claim 1, furthercomprising an adjuvant.
 4. The immunogenic composition of claim 1,wherein said immunogenic composition is devoid of an adjuvant.
 5. Theimmunogenic composition of claim 1, wherein said second attachment siteis capable of association to said first attachment site through at leastone covalent bond.
 6. The immunogenic composition of claim 1, whereinsaid composition further comprising a heterobifunctional cross-linker,which contains a functional group which can react with said firstattachment site and a further functional group which can react with saidsecond attachment site.
 7. The immunogenic composition of claim 6,wherein said heterobifunctional cross-linker is selected from the groupconsisting of SMPH, Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB,Sulfo-SMPB, Sulfo-SMCC, SVSB, and SLA.
 8. A pharmaceutical compositioncomprising: (a) the immunogenic composition of claim 1; and (b) anacceptable pharmaceutical carrier.